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HydraHarp 400
Picosecond Histogram
Accumulating Real–time Processor
User's Manual and Technical Data
Version 3.0.0.1
Time–Correlated
Single Photon Counting System
with USB Interface
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PicoQuant GmbH HydraHarp 400 Software V. 3.0.0.1
Disclaimer
PicoQuant GmbH disclaims all warranties with regard to the supplied software and documentation including all
implied warranties of merchantability and fitness for a particular purpose. In no case shall PicoQuant GmbH be
liable for any direct, indirect or consequential damages or any material or immaterial damages whatsoever
resulting from loss of data, time or profits; arising from use, inability to use, or performance of this software and
associated documentation.
License and Copyright Notice
With the HydraHarp 400 product you have purchased a license to use the HydraHarp software. You have not
purchased any other rights to the software itself. The software is protected by copyright and intellectual
property laws. You may not distribute the software to third parties or reverse engineer, decompile or
disassemble the software or part thereof. You may use and modify demo code to create your own software.
Original or modified demo code may be re–distributed, provided that the original disclaimer and copyright notes
are not removed from it. Copyright of this manual and on–line documentation belongs to PicoQuant GmbH. No
parts of it may be reproduced, translated or transferred to third parties without written permission of
PicoQuant GmbH.
HydraHarp is a registered trademark of PicoQuant GmbH. Other products and corporate names appearing in
this manual may or may not be registered trademarks or subject to copyrights of their respective owners.
PicoQuant GmbH claims no rights to any such trademarks. They are used here only for identification or
explanation and to the owner’s benefit, without intent to infringe.
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PicoQuant GmbH HydraHarp 400 Software V. 3.0.0.1
Table of Contents
1. Introduction...................................................................................................................................................... 5
2. Primer on Time–Correlated Single Photon Counting.......................................................................................6
2.1. Count Rates and Single Photon Statistics...............................................................................................7
2.2. Timing Resolution.................................................................................................................................... 8
2.3. Photon Counting Detectors......................................................................................................................9
2.3.1. Photomultiplier Tube (PMT)............................................................................................................. 9
2.3.2. Micro Channel Plate PMT (MCP).....................................................................................................9
2.3.3. Single Photon Avalanche Photo Diode (SPAD)............................................................................... 9
2.3.4. Other and Novel Photon Detectors................................................................................................10
2.4. Principles Behind the TCSPC Electronics.............................................................................................10
2.5. Further Reading..................................................................................................................................... 14
3. Hardware and Software Installation...............................................................................................................15
3.1. General Installation Notes – Read This First.........................................................................................15
3.2. Software Installation............................................................................................................................... 16
3.3. Device Installation.................................................................................................................................. 17
3.4. Installation Troubleshooting................................................................................................................... 18
3.5. Uninstalling the Software....................................................................................................................... 18
4. Software Overview......................................................................................................................................... 19
4.1. Starting the Program.............................................................................................................................. 19
4.2. The Main Window.................................................................................................................................. 19
4.3. The Toolbar........................................................................................................................................... 21
4.4. The Control Panel.................................................................................................................................. 22
4.5. The Axis Panel....................................................................................................................................... 23
4.6. The Trace Mapping Dialog..................................................................................................................... 24
4.7. Other Dialogs......................................................................................................................................... 24
5. Specific Measurement Tasks......................................................................................................................... 25
5.1. Setting Up the Input Channels............................................................................................................... 25
5.2. Setting Up and Running Interactive Measurements............................................................................... 29
5.3. Time Tagged Mode Measurements....................................................................................................... 30
5.3.1. System Requirements................................................................................................................... 30
5.3.2. T2 Mode......................................................................................................................................... 30
5.3.3. T3 Mode......................................................................................................................................... 31
5.3.4. Running a basic TTTR Mode Measurement..................................................................................32
5.3.5. External Markers............................................................................................................................ 33
5.3.6. Using TTTR Mode Data Files........................................................................................................ 33
5.3.7. TTTR Mode Measurements with Real–Time Correlation...............................................................34
5.4. Time–Resolved Excitation and Emission Spectra.................................................................................. 36
6. Controls and Commands Reference.............................................................................................................. 41
6.1. Main Window.........................................................................................................................................41
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6.2. Menus.................................................................................................................................................... 43
6.2.1. File Menu.......................................................................................................................................43
6.2.2. Edit Menu....................................................................................................................................... 45
6.2.3. View Menu.....................................................................................................................................46
6.2.4. Help Menu..................................................................................................................................... 47
6.3. Toolbar................................................................................................................................................... 48
6.4. Control Panel......................................................................................................................................... 50
6.4.1. Sync–Input..................................................................................................................................... 50
6.4.2. Inputs 1…4.................................................................................................................................... 50
6.4.3. Inputs 5…8.................................................................................................................................... 51
6.4.4. Acquisition..................................................................................................................................... 51
6.5. Axis Panel.............................................................................................................................................. 54
6.5.1. Time Axis Group............................................................................................................................54
6.5.2. Count Axis Group.......................................................................................................................... 54
6.6. Trace Mapping Dialog............................................................................................................................ 55
6.7. General Settings Dialog......................................................................................................................... 55
6.8. About HydraHarp… Dialog....................................................................................................................56
6.9. Title and Comment Editor...................................................................................................................... 56
6.10. Print Preview Dialog............................................................................................................................. 56
7. Problems, Tips & Tricks.................................................................................................................................58
7.1. PC Performance Issues......................................................................................................................... 58
7.2. USB interface......................................................................................................................................... 58
7.3. Histogram Artefacts............................................................................................................................... 58
7.4. Warming Up and Calibration..................................................................................................................59
7.5. Custom Programming of the HydraHarp................................................................................................59
7.6. Software Updates..................................................................................................................................59
7.7. Support and Bug Reports...................................................................................................................... 59
8. Appendix........................................................................................................................................................ 61
8.1. Warnings................................................................................................................................................ 61
8.2. Data File Formats.................................................................................................................................. 64
8.2.1. Interactive Histogramming Mode File Format................................................................................64
8.2.2. TTTR Mode File Format.................................................................................................................65
8.3. Hardware Technical Data...................................................................................................................... 66
8.3.1. Specifications................................................................................................................................. 66
8.3.2. Connectors.................................................................................................................................... 68
8.3.3. Indicators....................................................................................................................................... 69
8.4. Using the Software under Linux.............................................................................................................70
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1. Introduction
While fluorescence spectroscopic investigations are fairly common today, extracting additional temporal
information from molecules via laser induced fluorescence is a relatively new and much more powerful
technique. The temporal analysis can reveal information about the molecule not available from spectral data
alone. This is why lifetime analysis of laser induced fluorescence by means of Time–Correlated Single Photon
Counting (TCSPC) has gained importance over the recent years. The difference in the fluorescence decay
times of appropriate fluorescent dyes provides a powerful discrimination feature to distinguish molecules of
interest from background or other species. This has made the technique very interesting for sensitive analysis,
even down to the single molecule level.
The acquisition of fluorescence decay curves by means of TCSPC provides resolution and sensitivity that
cannot be achieved with other methods. In practice it is done by histogramming arrival times of individual
photons over many excitation and fluorescence cycles. The arrival times recorded in the histogram are relative
times between laser excitation and corresponding fluorescence photon arrival (start / stop times) ideally
resolved down to a few picoseconds. The resulting histogram represents the fluorescence decay. Although
fluorescence lifetime analysis is a main field of application for the HydraHarp, it is in no way restricted to this
task. Other important applications are e.g. Quantum Optics, Quantum Cryptography (QC) Time–Of–Flight
(TOF) and Optical Time Domain Reflectometry (OTDR) as well as any kind of coincidence correlation.
The HydraHarp 400 is a cutting edge TCSPC system with Universal Serial Bus (USB) interface. Its new
modular design provides a flexible number of input channels and allows innovative measurement approaches.
The timing circuits allow high measurement rates up to 12.5 million counts per second (Mcps) and provide a
time resolution of 1 ps. The modern USB interface provides high throughput as well as ‘plug and play’
installation. The input triggers are programmable for a wide range of input signals. All of them have a
programmable Constant Fraction Discriminator (CFD). These specifications qualify the HydraHarp 400 for use
with most common single photon detectors such as Single Photon Avalanche Diodes (SPADs) and
Photomultiplier Tube (PMT) modules (via preamplifier). The best time resolution is obtained by using Micro
Channel Plate PMTs (MCP–PMT) or modern SPAD detectors. The HydraHarp 400 is perfectly matched to
these detectors and the overall Instrument Response Function (IRF) can be as short as 30 ps FWHM.
Similarly, a wide range of excitation sources can be used, e.g. the interchangeable, easy–to–use LDH diode
laser series, as well as mode locked lasers such as a fs Ti:Sapphire laser. This permits lifetime measurements
down to under ten picoseconds with deconvolution, e.g. using the FluoFit Fluorescence Decay Fit Software.
The HydraHarp 400 can operate in various modes to adapt to different measurement needs. The standard
histogram mode performs real–time histogramming in on–board memory. Other modes are available via
software re-configuration. For instance, two different Time–Tagged–Time–Resolved (TTTR) modes allow
recording of each photon event on separate, independent channels, thereby providing unlimited flexibility in
off–line data analysis such as burst detection and time–gated or lifetime weighted Fluorescence Correlation
Spectroscopy (FCS) as well as picosecond coincidence correlation, using the individual photon arrival times.
The HydraHarp 400 is furthermore supported by a variety of accessories such as pre–amplifiers and an
optional hardware and software add–on allowing control of a monochromator from within the HydraHarp
software. The latter supports automated measurement of Time–Resolved Excitaion/Emission Spectra (TRES).
The HydraHarp software runs on all recent Windows PC platforms with Windows versions 7, 8 and 8.1,
including the x64 editions. It provides functions such as the setting of measurement parameters, display of
measurement results, loading and saving of measurement parameters and decay curves. Important
measurement characteristics such as count rate, count maximum and position, histogram width (FWHM) are
displayed continuously. Data can conveniently be exported via the clipboard, e.g. for immediate processing by
the FluoFit Fluorescence Decay Fit Software. An optional programming library (DLL) enables users to write
custom data acquisition programs for the HydraHarp in all modern programming environments.
For details on the Time–Correlated Single Photon Counting method, please read the next section as well as
our TechNote on TCSPC and consult the literature referenced at the end of section 2.4. Experienced users of
the method should be able to work with the HydraHarp straight away. Nevertheless, we recommend carefully
reading of the sections 3.2 and 3.3 on software and hardware installation to avoid damage. Later, the
comprehensive online–help function of the HydraHarp software will probably let the manual gather dust on the
shelf.
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2. Primer on Time–Correlated Single Photon Counting
In order to make use of a powerful analysis tool such as time–resolved fluorescence spectroscopy, one must
record the time dependent intensity profile of the emitted light. While in principle, one could attempt to record
the time decay profile of the signal from a single excitation / emission cycle, there are practical problems to
prevent such a simple solution in most cases. First of all, the decay to be recorded is very fast. Typical
fluorescence from organic fluorophores lasts only a few hundred picoseconds to some hundred nanoseconds.
In order to recover fluorescence lifetimes as short as e.g. 200 ps, one must be able to resolve the recorded
signal at least to such an extent, that the exponential decay is represented by some tens of sample points in
time. This means the transient recorder required would have to sample at e.g. 10 ps time steps. This is hard to
achieve with ordinary electronic transient recorders of reasonable dynamic range. Secondly, the light available
may be simply too weak to sample an analog time decay. Indeed the signal may consist of just single photons
per excitation / emission. This is typically the case for single molecule experiments or work with minute sample
volumes / concentrations. Then the discrete nature of the signal itself prohibits analog sampling. Even if one
has more than just a single molecule and some reserve to increase the excitation power to obtain more
fluorescence light, there will be limits, e.g. due to collection optic losses, spectral limits of detector sensitivity or
photo–bleaching at higher excitation power. The solution is Time–Correlated Single Photon Counting (TCSPC).
Since with periodic excitation (e.g. from a laser) it is possible to extend the data collection over multiple
excitation/emission cycles, one can reconstruct the single cycle decay profile from single photon events
collected over many cycles.
The TCSPC method is based on the repetitive, precisely timed registration of single photons of e.g. a
fluorescence signal. The reference for the timing is the corresponding excitation pulse. A single photon detector
such as a Photo Multiplier Tube (PMT) or a Single Photon Avalanche Photodiode (SPAD) is used to capture
the fluorescence photons. Provided that the probability of registering more than one photon per cycle is low,
the histogram of photon arrivals per time bin represents the time decay one would have obtained from a single
shot time–resolved analog recording. The precondition of single photon probability can (and must) be met by
attenuating the light level at the sample if necessary. If the single photon probability condition is met, there will
actually be no photons registered in many of the excitation cycles. The diagrams below illustrate how the
histogram is formed over multiple cycles.
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fluorescence photon
fluorescence photon
start-stop-time
start-stop-time
TCSPC
Histogram
laser pulse
many cycles do not
produce a photon
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The histogram is collected in a block of memory, where one memory cell holds the photon counts for one
corresponding time bin. These time bins are often (historically) referred to as time channels. In practice, the
registration of one photon involves the following steps: first, the time difference between the photon event and
the corresponding excitation pulse must be measured. For this purpose both signals are converted to electrical
signals. For the fluorescence photon this is done via the single photon detector mentioned before. For the
excitation pulse it may be done via another detector if there is no electrical sync signal supplied by the laser.
Obviously, all conversion to electrical pulses must preserve the precise timing of the signals as accurately as
possible. The actual time difference measurement is done by means of fast electronics which provide a digital
timing result. This digital timing result is then used to address the histogram memory so that each possible
timing value corresponds to one memory cell or histogram channel. Finally the addressed histogram cell is
incremented. All steps are carried out by fast electronics so that the processing time required for each photon
event is as short as possible. When sufficient counts have been collected, the histogram memory can be read
out. The histogram data can then be used for display and e.g. fluorescence lifetime calculation. In the following
we will expand on the various steps involved in the method and associated issues of importance.
2.1. Count Rates and Single Photon Statistics
It was already mentioned that it is necessary to maintain a low probability of registering more than one photon
per cycle. This was to guarantee that the histogram of photon arrivals represents the time decay one would
have obtained from a single shot time–resolved analog recording (The latter contains the information we are
looking for). The reason for this is briefly the following: Due to dead times of detector and electronics for at
least some tens of nanoseconds after a photon event, TCSPC systems are usually designed to register only
one photon per excitation / emission cycle. If now the number of photons occurring in one excitation cycle were
typically >1, the system would very often register the first photon but miss the following one or more. This
would lead to an over–representation of early photons in the histogram, an effect called ‘pile –up’. This leads to
distortions of the fluorescence decay, typically the fluorescence lifetime appearing shorter. It is therefore crucial
to keep the probability of cycles with more than one photon low.
To quantify this demand, one has to set acceptable error limits and apply some mathematical statistics. For
practical purposes one may use the following rule of thumb: In order to maintain single photon statistics, on
average only one in 20 to 100 excitation pulses should generate a count at the detector. In other words: the
average count rate at the detector should be at most 1 % to 5 % of the excitation rate. E.g. with the diode laser
PDL 800–B, pulsed at 80 MHz repetition rate, the average detector count rate should not exceed 4 Mcps. This
leads to another issue: the count rate the system (of both detector and electronics) can handle. Indeed 4 Mcps
may already be stretching the limits of some detectors and usually are beyond the capabilities of older TCSPC
systems. Nevertheless, one wants high count rates, in order to acquire fluorescence decay histograms quickly.
This may be of particular importance where dynamic lifetime changes or fast molecule transitions are to be
studied or where large numbers of lifetime samples must be collected (e.g. in 2D scanning configurations). This
is why high laser rates (such as 40 or 80 MHz from the PDL 800–B) are important. PMTs can safely handle
TCSPC count rates of up to 10 Mcps, standard (passively quenched) SPADs saturate at a few hundred kcps,
actively quenched SPADs may operate up to 5 Mcps but some types suffer resolution degradation when
operated too fast. Old NIM based TCSPC electronics can handle a maximum of 50 to 500 kcps, newer
integrated TCSPC boards may reach peak rates of 5 to 10 Mcps. With the HydraHarp 400, in each channel
average count rates of 6 Mcps and peak rates up to 12.5 Mcps can be collected. It is worth noting that the
photon arrival times are typically random so that there can be bursts of high count rate and periods of low count
rates. Bursts of photons may still exceed the average rate. This should be kept in mind when comparing count
rates considered here and elsewhere. The specifications for TCSPC systems may interpret their maximum
count rates differently in this respect. This is why another parameter, the so called dead –time is also of interest.
This quantity describes the time the system cannot register photons while it is processing a previous photon
event. The term is applicable both to detectors and electronics. Dead–time or insufficient throughput of the
electronics do not usually have a detrimental effect on the decay histogram or, more precisely, the lifetime to be
extracted from the latter, as long as single photon statistics are maintained. However, the photon losses
prolong the acquisition time or deteriorate the signal to noise ratio (SNR) if the acquisition time remains fixed. In
applications where the photon burst density must be evaluated (e.g. for molecule transition detection) dead–
times can be a problem. The HydraHarp 400 has an extremely short dead time of typically less than 80 ns,
imposing the smallest losses possible with instruments of comparable resolution today.
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2.2. Timing Resolution
The most critical component in terms of timing resolution in TCSPC measurements will usually be the detector.
However, as opposed to analog transient recording, the time resolution of TCSPC is not limited by the pulse
response of the detector. Only the timing accuracy of registering a photon determines the resolution. The
timing accuracy is limited by the timing uncertainty that the detector introduces in the conversion from a photon
to an electrical pulse. This timing error or uncertainty can be as much as ten times smaller than the detector's
pulse response. The timing uncertainties are usually quantified by specifying the rms error (standard deviation)
or the Full Width Half Maximum (FWHM) of the timing distribution or instrument response function (IRF). Note
that these two notations are related but not identical. Good, but also very expensive detectors, notably Micro
Channel Plate PMTs, can achieve timing uncertainties as small as 25 ps FWHM. Lower cost PMTs or SPADs
may introduce uncertainties of 50 to 500 ps FWHM.
The second most critical source of IRF broadening in fluorescence lifetime measurements with TCSPC is
usually the excitation source. While many laser sources can provide sufficiently short pulses, it is also
necessary to obtain an electrical timing reference signal (sync) for comparison with the fluorescence photon
signal. The type of sync signal available depends on the excitation source. With gain switched diode lasers
(e.g. PDL 800–B) a low jitter electrical sync signal is readily available. The sync signal used here is typically a
narrow negative pulse of −800 mV into 50 Ω (NIM standard). The sharp falling edge is synchronous with the
laser pulse (<3 ps rms jitter for the PDL 800–B). With other lasers (e.g. Ti:Sa) a second detector must be used
to derive a sync signal from the optical pulse train. This is commonly done with a fast photo diode (APD or PIN
diode). The light for this reference detector must be derived from the excitation laser beam e.g. by means of a
semi–transparent mirror. The reference detector must be chosen and set up carefully as it also contributes to
the overall timing error.
Another source of timing error is the timing jitter of the electronic components used for TCSPC. This is caused
by the finite rise / fall–time of the electrical signals used for the time measurement. At the trigger point of
comparators, logic gates etc., the amplitude noise (thermal noise, interference etc.) always present in these
signals is transformed to a corresponding timing error (phase noise). However, the contribution of the
electronics to the total timing error is usually small. For the HydraHarp it is typically 10 ps. Finally, it is always a
good idea to keep electrical noise pick-up low. This is why signal leads should be properly shielded coax
cables, and strong sources of electromagnetic interference should be kept away from the TCSPC detector and
electronics.
The contribution of the time spread introduced by the individual components of a TCSPC system to the total
IRF strongly depends on their relative magnitude. Strictly, the overall IRF is the convolution of all component
IRFs. An estimate of the overall IRF width, assuming independent noise sources, can be obtained from the
geometric sum of the individual components as an rms figure according to statistical error propagation laws:
Due to the squares in the sum, the total will be dominated by the largest component. It is therefore of little value
to improve a component that is already relatively good. If e.g. the detector has an IRF width of 200 ps FWHM,
shortening the laser pulse from 50 ps to 40 ps is practically of no effect. However, it is difficult to specify a
general lower limit on the fluorescence lifetime that can be measured by a given TCSPC instrument. In addition
to the instrument response function and noise, factors such as quantum yield, fluorophore concentration, and
decay kinetics will affect the measurement. However, as a limit, one can assume that under favourable
conditions lifetimes down to 1/10 of the IRF width (FWHM) can still be recovered via deconvolution.
A final time–resolution related issue worth noting is the channel width of the TCSPC histogram. As outlined
above, the analog electronic processing of the timing signals (detector, amplifiers, CFD etc.) creates a
continuous distribution around any true time value. In order to form a histogram, at some point the timing
results must be quantized. This is done by an Analog to Digital Converter (ADC) or an integrated Time–to–
Digital Converter (TDC). This quantization introduces another random error, if chosen too coarse. The
quantization step width (i.e. the resolution) must therefore be small compared with the continuous IRF. As a
minimum sampling frequency, from the information theoretical point of view, one would assume the Nyquist
frequency. That is, the signal should be sampled at least at twice the highest frequency contained in it. For
practical purposes one may wish to exceed this limit where possible, but there is usually little benefit in
sampling the histogram at resolutions much higher than 1/10 of the overall IRF width of the analog part of the
system.
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2.3. Photon Counting Detectors
2.3.1. Photomultiplier Tube (PMT)
A PMT consists of a light–sensitive photo cathode that generates electrons when exposed to light. These
electrons are directed onto a charged electrode called dynode. The collision of the electrons with the dynode
produces additional electrons. Since each electron that strikes the dynode causes several electrons to be
emitted, there is a multiplication effect. After further amplification by multiple dynodes, the electrons are
collected at the anode of the PMT and output as a current. The current is directly proportional to the intensity of
light striking the photo cathode. Because of the multiplicative effect of the dynode chain, the PMT is a photo
electron amplifier of high sensitivity and remarkably low noise. The high voltage driving the tube may be varied
to change the sensitivity of the PMT. Current PMTs have a wide dynamic range, i.e. they can also measure
relatively high levels of light. They are furthermore very fast, so that rapid successive events can be reliably
monitored. One photon on the photo–cathode can produce a short output pulse containing millions of
photoelectrons. PMTs can therefore be used as single photon detectors. In photon counting mode, individual
photons that strike the photo cathode of the PMT are registered. Each photon event gives rise to an electrical
pulse at the output. The number of pulses, or counts per second, is proportional to the light impinging upon the
PMT. As the number of photon events increase at higher light levels, it will become difficult to differentiate
between individual pulses and the photon counting detector will become non–linear. This usually occurs at
1..10 Mcps, dependent on the model. Similarly, in TCSPC applications, individual photon pulses may merge
into one as the count rate increases. This leads to pulse pile–up and distortions of the collected histograms.
The timing uncertainty between photon arrival and electrical output (transit time spread) is usually small
enough to permit time–resolved photon counting at a sub–nanosecond scale. In single photon counting mode
the tube is typically operated at a constant high voltage where the PMT is most sensitive.
PMTs usually operate between the blue and red regions of the visible spectrum, with greatest quantum
efficiency in the blue–green region, depending upon photo–cathode materials. Typical quantum efficiencies are
about 25 %. For spectroscopy experiments in the ultraviolet / visible / near infrared region of the spectrum, a
PMT is very well suited.
Because of noise from various sources in the tube, the output of the PMT may contain pulses that are not
related to the light input. These are referred to as dark counts. The detection system can to some extent reject
these spurious pulses by means of electronic discriminator circuitry. This discrimination is based on the
probability that some of the noise generated pulses (those from the dynodes) exhibit lower signal levels than
pulses from a true photon event. Thermal emission from the cathode that undergoes the full amplification
process can usually not be suppressed this way. In this case cooling of the detector is more helpful.
2.3.2. Micro Channel Plate PMT (MCP)
A Micro Channel Plate PMT consists of an array of glass capillaries (5–25 µm inner diameter) that are coated
on the inside with a electron–emissive material. The capillaries are biased at a high voltage. Like in the PMT,
an electron that strikes the inside wall of one of the capillaries creates an avalanche of secondary electrons.
This cascading effect creates a gain of 103 to 106 and produces a current pulse at the output. Due to the narrow
and well defined electron path inside the capillaries, the transit time spread of the output pulses is much
reduced compared to a normal PMT. The timing jitter of MCPs is therefore sufficiently small to perform time –
resolved photon counting on a picosecond scale, usually outperforming PMTs. Good but also expensive MCPs
can achieve timing uncertainties as low as 25 ps. Microchannel plates are in this respect the best match for the
HydraHarp 400 but they are limited in permitted count rate and provide lower sensitivity towards the red end of
the spectrum compared to suitably optimized SPADs.
2.3.3. Single Photon Avalanche Photo Diode (SPAD)
Avalanche Photo Diodes (APDs) are semiconductor devices, usually restricted to operation in the visible to
infrared part of the spectrum. Generally, APDs may be used for ultra–low light detection (optical powers <
1 pW), and can also be used as photon–counters in the so–called "Geiger" mode (biased slightly above the
breakdown voltage). In the case of the latter, a single photon may trigger an avalanche of about 108 carriers. In
this mode the device can be used as a detector for photon counting with very accurate timing of the photon
arrival. In this context they are also referred to as Single Photon Avalanche Photo Diodes (SPAD). Selected
and small devices may achieve timing accuracies down to 50 ps, but small devices are usually hard to align
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and difficult to focus into. Single–photon detection probabilities of up to approximately 50 % are possible. APDs
are often noisier than PMTs, but can have a greater quantum efficiency. Maximum quantum efficiencies
reported are about 70 %. The popular commercial devices from Excelitas, formerly Perkin Elmer (SPCM–AQR)
provide a timing accuracy of ~400 ps and are specified to have a quantum efficiency of 60 %. These modules
are thermoelectrically cooled for low dark count rate and deliver pre–shaped TTL pulses. They are the most
common detectors for applications where NIR sensitivity is important, e.g. single molecule detection. To
achieve the specified timing accuracy, exact focusing into the center of the active area is necessary. More
recent SPAD designs such as the PDM family from Micro Photon Devices have the benefit of much better
timing resolution and robustness, however, at the expense of a lower sensitivity at the red end of the spectrum.
2.3.4. Other and Novel Photon Detectors
The field of photon detectors is still evolving. Recent developments that are beginning to emerge as usable
products include so called silicon PMTs, Hybrid PMTs, superconducting nanowire detectors and APDs with
sufficient gain for single photon detection in analog mode. Each of these detectors have their specific benefits
and shortcomings. Only a very brief overview can be given here.
Silicon PMTs are essentially arrays of SPADs, all coupled to a common output. This has the benefit of creating
a large area detector that can even resolve photon numbers. The drawback is increased dark count rate and
relatively high afterpulsing.
Hybrid PMTs make use of a combination of a PMT front end followed by an APD structure. The benefits are
good timing and virtually zero afterpulsing while the need for very high voltage is a disadvantage. The hybrid
PMT modules of PicoQuant's PMA Hybrid series alleviate this problem by encapsulating the high voltage
supply, the detector, a peltier cooler and even a protective shutter in a compact housing.
Superconducting nanowires (typically made from NbN) can be used to create photon detectors with excellent
timing performance and high sensitivity reaching into the infrared. The shortcomings for practical purposes are
the extreme cooling requirements and the low fill factor of the wire structures, making it difficult to achieve good
collection efficiencies.
Another class of potentially interesting detectors are recently emerging APDs with very high gain. In
combination with an electronic amplifier they have been shown to detect single photons. As opposed to Geiger
mode, this avoids afterpulsing and allows very fast counting rates. The disadvantage is a high dark count rate,
currently way too high for any practical TCSPC application.
2.4. Principles Behind the TCSPC Electronics
For introductory purposes it is worth looking at the design of conventional TCSPC systems first. They consist of
the following building blocks:
The CFD is used to extract precise timing information from the electrical detector pulses that may vary in
amplitude. This way the overall system IRF may be tuned to become narrower and some of the random
background signal can be suppressed. The same could not be achieved with a simple threshold detector
(comparator). Especially with PMTs, constant fraction discrimination is very important, because their pulse
amplitudes vary significantly. Particularly pulses originating from random electrons generated at the dynodes of
the PMT can be suppressed because their avalanches had less time to amplify, and their corresponding output
pulses are small.
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The principle of a classical CFD is the comparison of the original detector signal with an amplified, inverted and
delayed version of itself. The signal derived from this comparison changes its polarity exactly when a constant
fraction of the detector pulse height is reached. The zero crossing point of this signal is therefore suitable to
derive a timing signal independent from the amplitude of the input pulse. In practice the comparison is done by
a summation. The timing is done by a subsequent threshold trigger of the sum signal using a settable level, the
so called zero cross trigger. Newer CFD designs achieve the same objective by differentiating the input signal
and triggering on the zero crossing of the differentiated signal. This is the case for the HydraHarp 400. This has
the benefit of adaption to different detector types without a need for changing physical delay lines.
Making the zero cross level adjustable (slightly above zero) allows to adapt to the noise levels in the given
signal, since otherwise an infinitely small signal could trigger the zero cross comparator. Typical CFDs
furthermore permit setting of a discriminator threshold that determines the lower limit the detector pulse
amplitude must pass. This is primarily used to suppress dynode noise from PMTs.
Similar as for the detector signal, the sync signal must be made available to the timing circuitry. Since the sync
pulses are usually of well defined amplitude and shape, a simple settable comparator (level trigger) is often
sufficient to adapt to different sync sources. Nevertheless, it can be valuable to have a CFD also on the sync
channel. A suitably designed CFD is also usable with pre–shaped signals and simply becomes a level trigger in
this case. This is the case also for the HydraHarp 400. Note that the input signals must have sufficiently fast
rise / fall times in the sub-nanosecond to 10 nanosecond range.
The signals from the two input discriminators / triggers are in conventional systems fed to a Time to Amplitude
Converter (TAC). This circuit is essentially a highly linear ramp generator that is started by one signal and
stopped by the other. The result is a voltage proportional to the time difference between the two signals. In
conventional systems the voltage obtained from the TAC is then fed to an Analog to Digital Converter (ADC)
which provides the digital timing value used to address the histogrammer. The ADC must be very fast in order
to keep the dead time of the system short. Furthermore it must guarantee a very good linearity (over the full
range as well as differentially). These are criteria difficult to meet simultaneously, particularly with ADCs of high
resolution (e.g. 12 bits) as is desirable for TCSPC over many histogram channels.
The histogrammer has to increment each histogram memory cell, whose digital address in the histogram
memory it receives from the ADC. This is commonly done by fast digital logic e.g. in the form of Field
Programmable Gate Arrays (FPGA) or a microprocessor. Since the histogram memory must also be available
for data readout, the histogrammer must occasionally stop processing incoming data. This prevents continuous
data collection. Sophisticated TCSPC systems solve this problem by switching between two or more memory
blocks, so that one is always available for incoming data.
While this section so far outlined the typical structure of conventional TCSPC systems, it is worth noting that
the design of the HydraHarp 400 is somewhat different. Today, it is state–of–the–art that the tasks
conventionally performed by TAC and ADC are carried out by a so called Time to Digital Converter (TDC).
These circuits allow not only picosecond timing but can also extend the measurable time span to virtually any
length by means of digital counters. The HydraHarp 400 does not use one such circuit but one for each input
channel and one for the SYNC input. They independently work on each input signal and provide picosecond
arrival times that then can be processed further, with a lot more options than in conventional TCSPC systems.
In the case of classical TCSPC, this processing consists of a subtraction of the two time figures and
histogramming of the differences. This is identical to the classical start–stop measurements of the conventional
TAC approach. The following figure exemplifies this for one detector channel (Start).
The full strength of the HydraHarp design is exploited by collecting the unprocessed independent arrival times
as a continuous data stream for more advanced analysis. Details on such advanced analysis can be found in
the literature. In this case the on–board memory is reconfigured as a large data buffer (FIFO) so that count rate
bursts and irregular data transfer are decoupled. This permits uninterrupted continuous data collection with
high throughput. This mode of operation is called Time–Tagged Time–Resolved (TTTR) mode. Details are
described in section 5.3.
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Forward and Reverse Start–Stop Mode
For simplicity it is most convenient to assume that the time delay measurement is directly causal, i.e. the laser
pulse causes a photon event and one measures the time delay between laser pulse and the subsequent
photon event. However, most conventional TCSPC systems need to give up this logical concept because of the
high repetition rates of the typical excitation lasers: Since the time measurement circuit cannot know in
advance whether there will be a fluorescence photon, it would have to start a time measurement upon each
laser pulse. Considering that conventionally conversion times are in the region of 0.3 to 2 ms, any excitation
rate in excess of 0.5 to 3 MHz would overrun the time measurement circuits. In fact they would most of the time
be busy with conversions that never complete, because there is no photon event at all in most cycles. By
reversing the start and stop signals in the time measurement, the conversion rates are only as high as the
actual photon rates generated by the fluorescent sample. These are (and must be) only about 1 to 5 % of the
excitation rate and can therefore be handled easily by the TAC / ADC. The consequence of this approach,
however, is that the times measured are not those between laser pulse and corresponding photon event, but
those between photon event and the next laser pulse. This still works (by software data reversing) but is
inconvenient in two ways:
1) Having to reverse the data leads to unpleasant relocation of the data displayed on screen when the
time resolution is changed.
2) Changing between slow and fast excitation sources requires reconnecting to different inputs, possibly
causing trouble when there is no CFD at one of the inputs.
The HydraHarp 400 is revolutionary in this respect, as it allows to work in forward start stop mode, even with
fast lasers. This is facilitated by two design features:
1) The independent operation of the time digitizers, and
2) a programmable divider in front of the sync input.
The latter allows to reduce the input rate so that the period is at least as long as the dead time. Internal logic
determines the sync period and re–calculates the sync signals that were divided out. It must be noted that this
only works with stable sync sources that provide a constant pulse–to–pulse period, but all fast laser sources
known today meet this requirement within an error band of a few picoseconds. Note: for slow sync sources (< 1
MHz) the sync divider must not be used (set to None). Similarly, the divider must not be used for coincidence
correlation measurements etc. since the sync rate has no influence on this kind of measurements. In summary:
The HydraHarp 400 is designed to always work in forward start–stop mode.
Experimental Setup for Fluorescence Decay Measurements with TCSPC
The figure below shows a typical setup for fluorescence lifetime measurements using one input channel of the
HydraHarp 400.
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The picosecond diode laser (PDL 800–B) is triggered by its internal oscillator (settable at 2.5, 5, 10, 20 and 40
MHz). The driver unit is physically separate from the actual laser head, which is attached via a flexible cable.
This permits to place the small laser head conveniently anywhere in the optical setup. The light pulses of
typically <70 ps FWHM, are directed toward the sample cuvette via appropriate optics. A neutral density filter
can be used to attenuate the light levels if necessary. Upon excitation, the fluorescent sample will emit light at a
longer wavelength than that of the excitation light. The fluorescence light is filtered out from scattered excitation
light by means of an optical cutoff filter (other configurations may utilize a monochromator here). Then it is
directed to the photon detector, again possibly via some appropriate collection optics, e.g. a microscope
objective or a lens.
As a photon detector the H5783 PMT from Hamamatsu is very convenient. It only needs a 12 V supply and
permits an instrument response width of <250 ps, allowing lifetime measurements even much shorter than this
via deconvolution. If a higher time resolution is required, the detector of choice is an MCP–PMT. The electrical
signal obtained from the detector (small negative pulses of typically −10 to −50 mV) is fed to the TCSPC
electronics via a preamplifier (e.g. PAM 105 from PicoQuant). This gives pulses of −100 to −500 mV. Cabling is
double shielded 50 Ω coax cable. If the detector is a SPAD module with TTL output (SPCM–AQR from
Excelitas, formerly Perkin Elmer) then an inverter / attenuator (SIA 400) must be inserted. Modern SPAD
devices like the PDM series from MPD directly provide negative timing signals.
The PDL 800–B laser driver readily provides the electric sync signal needed for the photon arrival time
measurement. This signal (a narrow negative pulse) is also fed to the TCSPC electronics via a high quality
50 Ω coax cable. When using the HydraHarp 400 in combination with the PDL 800–B, the sync pulse adapter
LTT 100 (brass cube with SMA connectors) or a 10 dB attenuator should be inserted directly at the sync output
of the laser driver. This reduces crosstalk into the relatively weak detector signals. If the laser does not provide
an electrical sync signal (e.g. Ti:Sa lasers), a sync detector (photo diode) such as the TDA 200 must be used.
The following figure shows TCSPC histograms obtained with this setup. Excitation source was a PDL 800–B
with a 470 nm laser head running at 20 MHz repetition rate. The narrower (blue) curve represents the system
IRF, here dominated by the detector (PMT). The other curve (dark red) is the fluorescence decay from a
solution of Coumarin 6 in ethanol, a fluorescent dye with fairly short fluorescence lifetime (~2.5 ns). The count
rate was adjusted to <1% of the laser rate to prevent pile–up. The plot in logarithmic scale shows the perfect
exponential nature of the decay curve, as one would expect it. Note that this is obtained even without a
deconvolution of the IRF.
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The approximate mono–exponential fluorescence lifetime can be obtained from a simple comparison of two
points in the curve with count rates in the ratio of 1 : 1/e (e.g. 100 000 : 36 788). In this particular experiment
this resulted in a lifetime estimate of 2.55 ns in good agreement with published results for this dye. For a
precise measurement one would perform a numerical exponential fit with IRF deconvolution (typically
implemented as an iterative reconvolution). This would result in slightly shorter lifetimes since the IRF broadens
the decay. Indeed one can measure lifetimes significantly smaller than the IRF with this method. Additionally,
the rms residue from the fit can be used to assess the quality of the fit and thereby the reliability of the lifetime
measurement. The FluoFit decay fit software package from PicoQuant provides this functionality.
2.5. Further Reading
1. O’Connor, D.V.O., Phillips, D.:
Time–correlated Single Photon Counting
Academic Press, London, 1984
2. Lakowicz, J. R.:
Principles of Fluorescence Spectroscopy, 3rd Edition
Springer, New York, 2006
3. Kapusta, P., Wahl, M., Erdmann, R. (Eds.):
Advanced Photon Counting - Applications, Methods, Instrumentation
Springer Series on Fluorescence, Vol. 15, Springer, 2015
ISBN 978-3-319-15635-4
4. Ortmann U., Wahl M., Kapusta P.:
Time-resolved fluorescence: Novel technical solutions.
Springer Series on Fluorescence, Vol.5, p.259-275 (2008)
5. Koberling F., Kraemer B., Buschmann V., Ruettinger S., Kapusta P., Patting M., Wahl M., Erdmann R.:
Recent advances in photon coincidence measurements for photon antibunching and full correlation
analysis. Proceedings of SPIE, Vol.7185, 71850Q (2009)
6. Wahl M., Rahn H.-J., Röhlicke T., Kell G., Nettels D., Hillger F., Schuler B., Erdmann R.:
Scalable time-correlated photon counting system with multiple independent input channels.
Review of Scientific Instruments, Vol.79, 123113 (2008)
7. O’Connor, D.V.O., Ware, W.R., Andre, J.C.:
Deconvolution of fluorescence decay curves. A critical comparison of techniques.
J. Phys. Chem. 83, 1333–1343, 1979
8. Patting M., Wahl M., Kapusta P., Erdmann R.:
Dead-time effects in TCSPC data analysis.
Proceedings of SPIE, Vol.6583, 658307 (2007)
9. http://www.picoquant.com/scientific/references
Bibliography listing all publications with work based on PicoQuant instruments
10. http://www.picoquant.com/products/category/tcspc-and-time-tagging-modules/hydraharp-400-
multichannel-picosecond-event-timer-tcspc-module#papers
Bibliography listing all publications with work based on the HydraHarp 400
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3. Hardware and Software Installation
3.1. General Installation Notes – Read This First
When handling the HydraHarp 400 system, be sure to avoid electrostatic discharges, especially when
connecting cables. Before connecting any signals, carefully study the maximum ratings given in section 8.3.1.
The HydraHarp software version 3.0.0.1 works with Windows 7, 8 and 8.11. In order to use the HydraHarp
software with the HydraHarp 400 hardware, a device driver must be installed. The HydraHarp 400 is a USB
‘plug and play’ device, this means the necessary resources and drivers are allocated automatically by the
operating system. Windows will automatically recognize when such a device is connected and load the
appropriate driver. The software setup for the HydraHarp 400 installs the driver so that upon connecting the
device it is readily available for the Windows plug&play device management. This requires that the software
setup is performed before connecting the device. Dependent on your version of Windows you may be
prompted to confirm the final driver installation upon connecting the device. Some older Windows versions
verbosely warn about drivers not being validated by Microsoft. You can safely ignore these warnings.
On some Windows versions you may need administrator status to perform the software setup and de –
installation. For the driver installation, it is needed in any case. Installing as Administrator has the benefit that
you can install the software for use by all users on that computer even if they have limited access rights. The
HydraHarp software will maintain individual settings for each user in the Windows registry. When switching
between users with sessions still running in the background you cannot run the HydraHarp software in multiple
sessions at the same time.
The HydraHarp software 3.0.0.1 is also suitable for 64-bit versions of Windows, where a suitable 64–bit device
driver will be installed. The HydraHarp software as such runs as a 32–bit application on WOW64.
Important Note for Software Version 2.0 and higher
Software versions 2.0 and higher require a firmware update of the HydraHarp device if it was purchased before
August 2012 and was not upgraded to new firmware since then. The HydraHarp software can perform this
update when it is first started.
The firmware update requirement has consequences that you must observe:
1. Once the update is performed you will no longer be able to use any HydraHarp software prior to version 2.0.
2. Custom software you may have written for file import of 1.x versions will require minor adaptions.
3. You will no longer be able to use custom software based on HHLib.dll prior to version 2.0.
4. Custom HHLib-based software you may have written for 1.x versions will require minor adaptions.
5. After the update you may need small CFD adjustments for any known good setup.
6. In case of a power failure or computer crash during the update the device may become inoperational.
7. Reverting to old firmware or repairing a disrupted update requires a return to factory and may incur costs.
Also note: When the firmware update has been performed the device must be switched off an on again in order
to become operational with the new software.
Important Note for Version 3.0 and higher
Software version 3.0 and higher uses a new file format with the file name extensions *.ptu for TTTR mode
files or *.phu for histogram data files. The idea of this new format is to place individual header data items not
in a strict file position and order but to “tag” the items by name, so that future additions do not harm existing
software. The new file format will therefore be “future proof” in terms of maintenance and compatibility across
new versions. The downside for now is that users who wrote their own data import routines for older formats
will need to adapt them to the new format one more time. This should be quite easy by means of demo code in
various programming languages provided as part of this release. For the first time this also includes a demo for
LabVIEW. For backward compatibility version 3.0 can still read (but not write) the previous format version 2.0.
1 Trademarks of Microsoft Inc.
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3.2. Software Installation
Before installing the new software you should (if applicable) back–up all HydraHarp data files you created in
any older HydraHarp directory (previous versions etc.) as well as any critical data on your PC. For a clean start
we also recommend to uninstall any older HydraHarp software.
The HydraHarp 400 setup files are normally provided on a CD supplied with your HydraHarp device. The
installer program file containing the complete distribution is setup.exe. If you received the software package
by download or any other means of electronic distribution, it will probably be packed in a ZIP–File. In this case
you can unzip this file to a temporary hard disk location of your choice and run the setup from there.
In order to use the HydraHarp software with the HydraHarp 400 hardware, a device driver must be installed.
The HydraHarp 400 is a USB ‘plug and play’ device, this means the necessary resources and drivers are
allocated automatically by the operating system. Windows will automatically recognize when such a device is
connected and load the appropriate driver. Note that the software setup for the HydraHarp 400 installs the
driver so that upon connecting the device it is readily available for the Windows plug&play device management.
It is recommended to perform the software setup before connecting the device. Dependent on your version of
Windows you may be prompted to confirm the final driver installation upon connecting the device. Some
windows versions verbosely warn about drivers not being validated by Microsoft. You can safely ignore these
warnings. Starting with HydraHarp software version 2.1 the driver package will appear as a separate software
installation that can be uninstalled like any other software through the standard control panel mechanisms.
On some Windows versions you will need administrator status to perform the software setup. For the driver
installation you need it in any case. You can run the setup program directly from the CD or from the hard disk
location where you unpacked the electronic distribution.
To perform the installation, insert the installation disk in your CD drive. Open the CD location either
from the Windows desktop or via the Windows Explorer. If you unpacked the setup file to a hard disk
location, open that location. Run setup.exe. The setup program will guide you through the
installation process step by step.
When asked for a destination folder for the new software, please accept the default path or select another
according to your program storage policies. This is where the HydraHarp files will be installed. To avoid
confusion, make sure not to specify the path of an older HydraHarp version that you have not uninstalled or
that of any other program on your PC.
The default location is: \Program Files\PicoQuant\HydraHarp400v30.
Setup will also create a dedicated "program folder" for the new HydraHarp 400 software that will later appear in
the Start Menu. You can accept the default folder name or select another according to your own naming
policies. However, you should make sure not to specify the folder name of an older HydraHarp version that you
have not uninstalled nor the dedicated folder of any other program.
The setup program will install the following files in the chosen destination folder:
HydraHarp.exe the main executable program
HydraHarp.chm the HydraHarp compiled HTML–Help file
Manual.pdf the manual in Adobe PDF format
Readme.txt a text file with license information
Readme.txt a text file with program information
pquwstub.dll a helper DLL for operation under Linux with Wine
unins000.exe the uninstaller program file
unins000.dat the uninstaller information file
Setup will also create a subdirectory \filedemo which contains demo source code for access to
HydraHarp data files in various programming languages. Another folder \sampledata will be created with
samples of HydraHarp data files. Other necessary files such as setup information and the device driver will be
installed in the standard places in your Windows directory tree.
Setup will also install a File Info shell extension that you can use to inspect individual header items of a *.ptu
or *.phu file. This includes the measurement mode. Just right-click on the file in Windows explorer and select
Properties. Then look at the tab PQ File Info and the tab PQ File Comment.
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After the installation the HydraHarp software should be available in the Windows Start Menu under Programs |
PicoQuant – HydraHarp 400 v3.0 (or the folder name you chose during setup). However, before running the
software, please proceed to the hardware installation step.
3.3. Device Installation
Make sure to avoid electrostatic discharge when handling the HydraHarp system, especially when connecting
cables. Note that PMT detectors operate with high voltage that may discharge through the signal cable. Make
sure such detectors are switched off and fully discharged before connecting them.
Dependent on the model of the HydraHarp device you purchased it has a USB 2.0 or USB 3.0 interface.
Consult your PC manual as to whether and where it provides high speed USB 2.0 or super speed USB 3.0
connectors. The latter are typically blue. The minimum requirement is a USB 2.0 connection. The HydraHarp
system will NOT work through USB 1.x. All current PCs should provide at least USB 2.0 connectivity. Most
recent PCs will provide USB 3.0 out of the box. If you purchased a HydraHarp with USB 3.0 interface but the
PC has no USB 3.0 support you can install a USB 3.0 adaptor card. It is also possible to run a USB 3.0
HydraHarp device via USB 2.0 but performance will be poor. Use a proper USB 3.0 connection whenever
possible.
Always use a quality USB cable rated for the chosen USB speed. The cable length must not exceed 5 metres
(~16 ft). For best reliability we recommended to use the provided cable of 3 metres length. Note that the USB
specification does not allow cable extensions other than dedicated active extension cables or hubs. The
HydraHarp device should work flawlessly through suitable hubs. This is also a valid way of extending the
maximum cable length. After a hub, another cable of up to 5 metres is allowed. Note, however, that hubs may
lower the data throughput. For the same reasons it is recommended not to connect other bandwidth
demanding devices to the same hub.
The inputs for the detector / sync signals are SMA connectors located on the front panel of the HydraHarp 400,
labelled IN and SYNC IN. In case of time resolved fluorescence experiments with a pulsed excitation source,
the sync signal must be connected to the SYNC IN input and the detector signal must be connected to an IN
port. If coincidence correlation experiments between two (or more) detector signals are to be carried out, you
need to decide whether you will be using on–board histogramming or TTTR mode. In the case of on–board
histogramming connect one detector to the SYNC input and one or more to the regular IN ports. Coincidence
timing and histogramming will always be with respect to the SYNC input. In TTTR mode it is possible to
determine the relative timing between all inputs but this requires off–line data analysis (see section 5.3).
All inputs are terminated with 50 Ω internally. Note that they need negative going input signals. Use quality
50 Ω coax leads with appropriate connectors. For interfacing to BNC connectors use standard adaptors.
Carefully screw on the SMA connectors for sync and detector until they are moderately tight. Do not use
wrenches. Connect the cable ends to the appropriate signal sources (50 Ω) in your experimental setup. The
HydraHarp 400 inputs accept negative pulses going from 0 V to max. –1 V. Both inputs should be operated
with similar pulse amplitudes to minimize crosstalk. The optimum range is –100 mV to –500 mV. Below this
range you may pick up noise, above there may be crosstalk. Most PMT and MCP detectors will require a pre–
amplifier to reach enough signal level. TTL–SPAD–detectors (e.g. Excelitas SPCM–AQR) must be connected
through a pulse inverter (PicoQuant SIA 400). Weak PMT detectors should be connected through a 20 dB high
speed pre–amplifier. MCP–PMT detectors should be connected through an amplifier with slightly higher gain.
Suitable devices are available from PicoQuant. Be sure to switch the high voltage supply of PMTs off and allow
their electrodes to discharge before connecting them. Their high voltage charge may damage the pre–amplifier.
Observe the allowed maximum ratings for the input signal levels. If you are not sure what signals your setup
delivers, use a fast oscilloscope to check the signal level and shape before connecting them to the HydraHarp.
All signals should have rise/fall times of no more than 2 ns. Slower signals may not be seen by the HydraHarp
and will certainly degrade timing accuracy.
When using the HydraHarp 400 in combination with the PDL 800–B, the sync pulses from the laser driver
should be attenuated by 10 dB to 15 dB to fall in the optimum range for smallest crosstalk.
Do not connect anything other than dedicated hardware to the other HydraHarp 400 connectors. They are
provided for hardware expansion (notably experiment control) and must not be used otherwise. See
section 8.3.2 for pin assignments. It is recommended to start instrument setup without anything connected to
the control ports.
After connecting the HydraHarp to the PC via USB for the first time, Windows should detect the device and
perform the final driver installation. Note that the software setup for the HydraHarp 400 pre–installs the driver.
Dependent on your version of Windows you may be prompted to confirm the final driver installation. Some
older windows versions verbosely warn about drivers not being validated by Microsoft. You can safely ignore
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these warnings. After driver installation Windows should report the device as ready to use. If you wish to verify
correct installation you can check the presence of 'HydraHarp 400' as a USB device in the Windows Device
Manager. For troubleshooting see the next section.
3.4. Installation Troubleshooting
After completion of the software setup and device installation the HydraHarp device should be listed in the
Windows Device Manager under the USB tree. Right click My Computer > left click Properties > Device
Manager to check if the device is free of conflicts and / or if the device driver is installed correctly. Under USB
look for a device named HydraHarp 400 and inspect its Properties.
You can also use the Windows system information facilities (Start > Run > msinfo32). In the System
Information utility inspect Software environment > System Drivers to check if the HydraHarp device driver
PQUSB.SYS (PQUSB64.SYS on x64) is correctly installed and running.
You can also repeat the software installation if necessary. Disconnect the device, uninstall the software and
repeat the setup procedure. Make sure the software is not installed in multiple places. If this does not resolve
the problem, try a different computer. If problems persist, see section 7 for support.
3.5. Uninstalling the Software
Before uninstalling the HydraHarp software you should back–up all valuable data files you might have created
in the HydraHarp installation directory.
Do not manually delete any program files from the installation folder as it will render a clean uninstall
impossible. Also do not delete any driver files manually.
To uninstall the HydraHarp software from your PC you may need administrator rights (dep. on Windows
version and security settings). Go to Control Panel > Add/Remove Software or Programs and select
PicoQuant – HydraHarp 400 vx.x for un–installation. This will remove all files that were installed by the
HydraHarp setup program but not the user data that may have been stored. If there was user data in the
program folders or any subfolders, these will not be deleted by the uninstall program. If intended, you need to
delete these files or folders manually. Nevertheless, it is recommended to back–up valuable measurement data
before uninstalling the software.
Note that un–installation of the data acquisition software does not uninstall the device driver. Do not delete the
driver software from within Device Manager. Starting with HydraHarp software version 2.1 the driver package
appears as a separate software installation that can be uninstalled like any other software through the standard
Windows Control Panel mechanisms (Add/Remove Software or Programs). In the list of installed software look
for items called Windwos Driver Package - PicoQuant (PQUSB). Note, however, that the driver package may
still be needed by other PicoQuant devices. Uninstall it only if you are sure it is no longer required.
Note also that un–installation of the data acquisition software does not automatically uninstall the PQ File Info
shell extension. It can be un-installed as a separate item through the standard Windows Control Panel
mechanisms.
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4. Software Overview
4.1. Starting the Program
After correct installation the Start Menu Programs contains a shortcut to the HydraHarp software. To start the
HydraHarp software select PicoQuant – HydraHarp 400 v3.0.0.1. Note that after switching the HydraHarp on,
you need to allow a warm–up period of at least 20 minutes before using the instrument for serious
measurements. You can use this time for set–up and preliminary measurements.
If the HydraHarp software cannot find the device (or if there are driver problems) it will display a notification
message, but it can still start. Device dependent toolbar buttons and functions of the program will then remain
disabled. This allows you to use the software without the HydraHarp hardware, e.g. to view or print files on
another computer.
If the HydraHarp 400 is correctly installed and there are still device related errors, you can use the Windows
Device Manager for troubleshooting (see the corresponding section above). If problems cannot be resolved,
see section 7 for support. If possible, try the device on another computer.
For regular use of the HydraHarp you may want to create an icon for it on your Windows desktop. If you missed
this during software setup you can do it any tim later via a right mouse click on the start menu entry ( send to >
Desktop – create shortcut). Alternatively use the Windows Explorer to locate the file HydraHarp.exe in the
directory you selected for installation and drag the icon onto the desktop. This will create a link to the
HydraHarp executable file.
You can also start up the HydraHarp software directly from a HydraHarp histogram file by double–clicking on
the file or dragging it onto the HydraHarp icon.
4.2. The Main Window
The HydraHarp software provides a measurement control interface to the HydraHarp hardware and an online
histogram display. The HydraHarp main window accommodates the histogram display area.
Above the display area is the toolbar. Here you can access frequently used commands by simple mouse click.
Above the toolbar is the menu bar for access to additional commands. At the bottom of the histogram display
area is a set of ‘panel meters’ showing count rates, count sums, and histogram peak characteristics. These will
be updated continuously, some only when a measurement is in progress. Note the selector at the right of the
panel meters. This selects the channel the meters are displaying. Instead of a single channel you can also
select Sum, which then displays the summed rates from all channels. The panel meters can be enlarged by
double–clicking, which is useful for optical alignment etc. when the PC is at some distance.
In the top center of the display area a title line is shown. This can be double–clicked to edit the title. When
editing the title, note that only the first line will appear in the display. The remaining lines are meant to be used
as a file comment. All lines will be stored with the file data, but the maximum total is 256 characters.
The main window is resizeable and the actual histogram display will adapt its size accordingly. If you make the
window smaller than the minimum histogram display, two scrollbars will permit access to hidden window areas.
Note that the actual size of the main window depends on the system font selected. It will scale to the size of the
current system font. Screen resolutions under 800x600 are not suitable for serious work with the software.
Note that the position and size of the main window on the screen will be stored in the Windows registry and
retrieved during the next program start. The registry settings are kept separately for each user, provided
he / she is logged on with a unique user name. Consult the section 6.1 for further main window command
descriptions. Toolbar, Menus, panel meters etc. will be explained in the next sections.
At the very bottom of the main window there is a status bar. The leftmost area of the status bar describes
actions of menu items as you navigate through menus. Similarly it shows messages that describe the actions
of toolbar buttons. The second status bar area from the left shows the current measurement status of the
HydraHarp. The rightmost area of the status bar indicates if the <Caps> and <NumLock> keys are latched
down.
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When the HydraHarp software is running with functional hardware it continuously collects
information about the input signals and the current acquisition settings. If these settings together
with the input rates indicate possible errors, the software will activate a warning icon in the status
bar. The warning icon can be clicked to display a list of current warnings together with a brief
explanation of each warning (see also section 8.1).
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4.3. The Toolbar
The toolbar is displayed across the top of the HydraHarp main window, below the menu bar. The toolbar
provides quick mouse access to frequently used commands and tools. Note that some buttons may be grayed
out (disabled) dependent on the installed software components and / or the state of the hardware.
To hide or display the Toolbar, choose Toolbar from the View menu (<Alt>+V T). The toolbar is capable of
‘docking’, i.e. you can move it to another edge of your window or even have it displayed as a separate window.
The following table explains the individual buttons.
Click… to…
open a blank histogram with default control panel settings
open an existing histogram file. Displays the Open dialog box, in which you
can locate and open the desired file.
save the current histogram data with its current name. If you have not named
the file, the Save As… dialog box is displayed.
copy the currently displayed curves to the clipboard (ASCII export).
print the currently displayed histogram curves.
display the About… window. This is where you can determine the version of
your HydraHarp software and hardware. Also provides links for updates etc.
activate context help.
launch the axis panel.
launch the data cursor dialog.
calibrate the time measurement hardware
start measurement based on current HydraHarp control panel settings
stop measurement and histogram accumulation.
launch the HydraHarp control panel.
launch the trace mapping dialog.
launch the TTTR mode dialog.
Launch the TTTR mode real–time correlator dialog.
launch the dialog for monochromator control and TRES setup.
launch the general settings dialog.
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4.4. The Control Panel
The HydraHarp control panel is a dialog box for setting the parameters for hardware adjustment and data
acquisition. It is implemented as a ‘non–modal’ dialog box, i.e. it does not have to be closed before the main
window can continue. This way you can make changes to your settings in the control panel and watch their
effect on a running measurement in the main window immediately. Nevertheless, you may close the control
panel and restore it at any time by clicking the control panel button on the toolbar or pressing <Alt>+C.
The control panel consists of several pages (tabbed sheets) containing groups of edit boxes and other controls
for related parameters. These pages and their respective controls are:
Sync–Input
Zero Cross edit box and spin control
Discriminator Level edit box and spin control
Offset edit box and spin control
Sync Divider edit box and spin control
Inputs 1…4
Zero Cross edit box and spin control
Discriminator Level edit box and spin control
Offset edit box and spin control
All=Ch1, ZeroCr. tick box
All=Ch1, Discr. tick box
All=Ch1, Offset tick box
Controls for channels that do not exist are gray.
Inputs 5…8
This page only exists when more than 4 input channels are installed. It has the same controls as the
page for inputs 1..4 and follows the same logic of operation.
Acquisition
Resolution edit box and spin control
Time edit box and spin control
Offset edit box and spin control
Trc/Block trace colour indicator,
edit box and spin control
Restart check box
Stop on Overflow check box
Mode drop down selection box
Stop At edit box and spin control
Edit boxes are for keyboard entry. The values must be confirmed with either the <Enter> key or the Apply
button. The spin controls can be used to increment or decrement the edit box values. In this case the changes
take effect immediately without need for <Enter> or Apply. Check boxes have their denoted effect when the
tick is shown. They can be toggled with a mouse click. Groups of radio buttons are like check boxes but
mutually exclusive.
Note that the settings of the control panel as well as the positions of control panel and main window on the
screen will be stored in the Windows registry and retrieved during the next program start. The registry settings
are kept separately per user. When a HydraHarp data file is loaded, the control panel settings will change
according to the settings in that file.
The individual control panel items are discussed in the section 6.4 Control Panel in the Controls and
Commands Reference.
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4.5. The Axis Panel
The axis settings panel is a dialog box for setting the axis range for the histogram display in the main window. It
is implemented as a ‘non–modal’ dialog box, i.e. it does not have to be closed before the main window can
continue. This way you can make changes in the axis panel and watch their effect in the main window
immediately. Nevertheless, you may close the axis panel and restore it at any time by clicking the axis panel
button on the toolbar or pressing <Alt>+A. The panel will also open if you double–click the axes in the main
window.
The axis panel consists of two groups containing edit boxes and other controls for related parameters. These
groups and their respective controls are:
Time Axis
Minimum edit box and spin control
Maximum edit box and spin control
Count Axis
Minimum edit box and spin control
Maximum edit box and spin control
Linear radio button
Logarithmic radio button
The edit boxes are for keyboard entry. The values must be confirmed with either the <Enter>–key or the
Apply–button. The spin controls can be used to increment or decrement the edit box values. In this case the
changes take effect immediately without need for <Enter> or Apply. Check boxes have their denoted effect
when the tick is shown. They can be toggled with a mouse click. Groups of radio buttons are like check boxes
but mutually exclusive. They work like on an old radio: one that was pressed pops out when another one is
pressed.
Note that the settings of the axis panel as well as the positions of the panel on the screen will be stored in the
Windows registry and retrieved during the next program start. The registry settings are kept separately per
user.
The individual axis panel items are discussed in the section 6.5. “Axis Panel“ in the Controls and Commands
Reference.
To begin, use logarithmic display to make sure even weak signals can be seen.
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4.6. The Trace Mapping Dialog
The HydraHarp software can measure histograms in up to 512 memory blocks. Out of these up to 8 curves can
be displayed. The Trace Mapping dialog is used to select the curves to display. It also allows to view curve
details and cleanup. You can use the Trace Map button on the Toolbar or click the curve colour indicator in the
control panel to launch the dialog.
In the Trace Mapping dialog you can tick the individual boxes ‘Show’ to display a curve. Select the number of
the memory block you wish to map the individual display curve to. Choose the active memory block you wish to
use for the next measurement in the HydraHarp control panel with the Map To edit box and spin control.
The dialog also provides some statistics on each curve (central matrix of figures). Resolution is the channel
width (bin width) of the histogram in picoseconds. Next is the Max. Count, the count in the highest point of the
curve. The column At Time shows the time corresponding to the Max. Count channel. Leftmost there is the Full
Width Half Maximum (FWHM) of the curve peak (usually for IRF traces).
There are also some buttons for frequently required actions: The button 'view' can be clicked to see more
detailed curve information such as time of recording, acquisition settings and count rates. The button All can be
clicked to tick all traces as shown. The button 0..7 can be clicked to set the default mapping of trace 0..7 to
block 0..7. The Clear buttons (trash cans) can be clicked to delete the contents of individual blocks.
Note that the Trace Mapping Dialog is non–modal. This means the dialog can remain open while a
measurement is in progress, so that adjustments can be made under immediate visual control, similar to the
operation of the control panel. Note also that a measurement can be running in a block that is not mapped or
shown.
The individual axis panel items are discussed in the section 6.6. “Trace Mapping“ in the Controls and
Commands Reference.
4.7. Other Dialogs
In order to keep this manual readable, the dialogs described here are limited to the most important ones the
reader should know about before starting practical work with the software. Some more dialogs will be described
implicitly in the following sections in the context of specific measurement tasks. For information on all other
dialogs the user is kindly requested to refer to the controls and commands reference (section 6) or consult the
on–line help facility of the software. Pressing F1 in any given dialog will provide help on that dialog.
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5. Specific Measurement Tasks
5.1. Setting Up the Input Channels
This section provides help and instructions for the first basic steps of setting up the instrument. However, if you
are running TCSPC measurements for the first time we strongly recommend you read our TechNote on
TCSPC first. The document is on your distribution CD. This will help you getting started more quickly.
In order to acquire any data, the input channels and the SYNC input of the HydraHarp 400 must be set to
match their electrical input signals. The HydraHarp input channels are designed identically, all with a Constant
Fraction Discriminator (CFD). They can handle an input pulse peak range from 0 to −1 V for regular operation.
The absolute maximum rating (beyond which damage may occur) is ±1.5 V.
In case of coincidence correlation experiments between two or more detectors, the channels are typically used
for one detector each and data is collected in T2 mode. The SYNC is then usually not required. If a quick
visualization of coincidence is required, it is also possible to use histogramming mode. In that case, one
detector is connected to the SYNC input. Coincidence histograms will be collected for each input channel with
respect to the SYNC input. In time–resolved fluorescence measurements with a pulsed excitation source
(typically a laser) the SYNC input receives a sync signal from the laser. Here we focus on the latter, more
common case. Perform the following steps to set up the input discriminators.
Using the Panel Meters
At the bottom of the main window you find a set of panel meters. These are very important during set–up. The
meters showing units of cps (counts per second) in their title are rate meters. The leftmost rate meter shows
the Sync input rate. The next meter shows the channel input rate. Note the selector at the right of the panel
meters. This selects the channel the rate meters (except Sync) are referring to. Select the channel you are
currently setting up. The HydraHarp software enumerates the physical input channels as logical input channels
1 to N, where N depends onthe number of installed TDC modules. The mapping of physical to logical channels
is obtained by counting as follows: start form the leftmost TDC module and count top to bottom within each
TDC module. At the time of factory assembly this mapping will be marked by plastic labels at each IN
connector.
The other meters show the histogramming rate, the total count in the histogram, the maximum (peak) count
and the position of the maximum. They all depend on the input selector.
Note that the Input selector also has a selection option Sum. In this case the meters are fed from the sum of all
input channels. Also note that the internal rate counters have a gate time of 100 ms. Very low rates can
therefore not be measured very accurately and the meter reading may fluctuate between two values.
Setting up the SYNC Input
For typical fluorescence decay measurements, the HydraHarp needs an electrical SYNC signal from the light
source. The PDL 800 family of diode lasers from PicoQuant provides this signal directly. If the laser does not
provide an electrical sync signal (e.g. Ti:Sa lasers), a SYNC detector (photo diode) such as the TDA 200 must
be used. The SYNC signal must consist of pulses with falling active edge. The pulse edges must be steep
(2 ns rise⁄fall time or faster). For example, a NIM type signal is appropriate. This is a steep negative pulse
(0.5 ns … 1 ns wide) of typically −800 mV into 50 Ω. The HydraHarp can actually handle −1 V but amplitudes in
excess of −800 mV may cause excess interference and crosstalk between the input channels. Amplitudes
around −200 mV … −300 mV are best in terms of timing accuracy and lowest histogram ripple. Another rule of
thumb for lowest signal distortion is to use signals of similar amplitude on all channels. SMA in-line attenuators
of suitable bandwidth can be used to adjust this. The adjustable CFD level is restricted to −1000 mV to 0 mV.
Initially you should set it to half of the SYNC pulse amplitude. The zero cross setting should be set to 10 mV
initially.
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Unless you are sure what kind of signal your SYNC source delivers, use a fast oscilloscope (50 Ω input) to
check the pulse shape, polarity and amplitude. The leading edge should be steep (2 ns fall time or faster),
there should also be no excessive overshoot or ringing. The pulse width should be 0.5 ns … 30 ns. The upper
limit is not critical, however, the pulse width should be as small as necessary to fit in half the SYNC period.
If the signal is satisfactory, connect the source to the SYNC input and start the HydraHarp software if it is not
yet running. A detector signal (Channels 1..8) is not required at this point but it does no harm if it is also
connected. Open the HydraHarp control panel. Leftmost, there is a tab with a group of controls for the SYNC
input. Select the corresponding tab and find the edit box and spin control for the CFD level. The level should be
set to a value around half of the amplitude of the SYNC pulses. Note that the control panel shows absolute
numbers from 0 to 1000 mV despite the real signals are negative going. Next, set the Zero Cross level to
10 mV. Then look at the Sync Divider. It must be set so that the sync rate divided by the shown divider value
does not exceed 12.5 MHz, e.g.,for a diode laser with 80 MHz repetition rate it must be set to 8. For a slow
source such as a flash lamp it must be set to None.
If the SYNC source is active, the Sync rate will now be displayed at the bottom left rate meter in the HydraHarp
main window. Note that the rate meter internally corrects for the chosen divider setting, so that the meter
always shows the undivided input rate. The meter display should therefore match the rate delivered from the
source. The rate meter will be refreshed every 0.1 to 1 seconds, determined by the value in the General
Settings Dialog. The Sync rate should be displayed very accurately unless it is very low. Large fluctuations or
occasional zeros indicate an incorrect discriminator level setting or an unstable Sync signal. Try to vary the
discriminator level to obtain a stable Sync rate display. If the rate is stable and as expected, you can proceed to
the set–up of the other input. A last fine tuning of the discriminator and zero cross level can be done when the
detector inputs are up and running.
Setting up the Photon Detector Inputs (Channel 1…8)
Dependent on the number of installed TDC modules your HydraHarp may have 2, 4, 6 or 8 input channels. As
noted before, the HydraHarp 400 input channels are designed electrically identical, each with a CFD. The
signals must consist of pulses with falling active edge. The pulse edges must be steep (2 ns rise / fall time or
faster). The inputs can handle a voltage range of 0 to −1 V. Note that some SPAD–detectors (e.g. SPCM–
AQR) deliver positive pulses of ~3 V (TTL) and must therefore be connected through a pulse inverter with
attenuation (PicoQuant SIA 400). Connecting them directly will cause damage. PMTs should be connected
through a preamplifier (10 dB … 20 dB). MCP–PMT detectors should be connected through an amplifier with
slightly higher gain. All accessories are available from PicoQuant. Be sure to switch the high voltage supply of
PMTs off and allow their electrodes to discharge before connecting/disconnecting them. Their high voltage
charge may damage the preamplifier. Observe the allowed input signal levels including those of the pre–
amplifier. Again, in a new experimental setup, to be absolutely sure, please check your detector pulses as well
as the preamp output with a fast oscilloscope. Start timing is on the leading edge, so it should be steep.
Ringing and overshoot should be as small as possible. Do not over–illuminate the detector to avoid damage.
If the signals are appropriate, connect a detector at channel 1. Starting with only one detector makes the first
steps easier. Preliminary adjustments can actually be done with uncorrelated light, e.g. daylight. For safety of
your detector, use strongly attenuated light, even when the detector is off. Start the HydraHarp software and
open the Control Panel. In the Control Panel section for the chosen Channel, initially set the CFD Discriminator
Level to 30 mV and the CFD Zero Cross level to 10 mV.
Using the input selector at the right of the rate meters, select the channel you are currently using. The rate
meter next to the sync rate meter will now show the input rate at that channel. Make sure that there is actually a
signal coming in (some strongly attenuated light on the detector) and try to adjust the CFD discriminator level.
To monitor this, watch the count rate meter. The adjustment range is 0 mV … 1000 mV for the CFD
discriminator level and 0 mV … 40 mV for the CFD zero cross level. Note that a precise tuning of the CFD
settings is only critical with PMT or MCP–PMT detectors. For such detectors the initial setting of the
discriminator level of 30 mV should be approximately right to suppress any electrical noise. If you lower it, you
should see an increase in count rate (probably mostly noise). If you raise it you will see a reduced rate.Thus,
you should stay just slightly above the noise level. If you still see millions of counts per second at levels over
50 mV, reduce the detector illumination. Your detector may be at risk at such high count rates. You can then
also try out responsivity to illumination changes. The Zero Cross level is not yet meaningful. It can be adjusted
for optimum timing later. For SPAD detectors that typically deliver pre–shaped pulses of constant amplitude the
CFD set–up is very simple. Just set the level to approximately half of the pulse amplitude. Again, the Zero
Cross level is not yet critical. It can be adjusted for optimum timing later.
To actually collect histograms, select a measurement range large enough (determined by the chosen
resolution) and a display range to cover your SYNC interval (i.e. 1 / sync) if possible. Set Offset = 0 and
StopAt = 4,294,967,295. Start a measurement in oscilloscope mode with e.g. 1 second acquisition time (see
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the next section for instructions). If there are counts coming in, you can try to set an upper limit of the count
axis so that the histogram is scaled for best viewing. Start with logarithmic display so that weak signals can be
seen.
If so far you used uncorrelated light, you may now want to move to time–correlated measurements. With a
detector signal e.g. laser induced, or a ‘fake’ electrically derived from the laser SYNC, you can try to obtain a
histogram that should be a narrow peak, as opposed to the flat distribution of uncorrelated light. This requires
an experimental setup that delivers the signals at the two input channels with a more or less constant relative
delay. For the HydraHarp in histogramming mode, this delay must be such that the Channel 1..8 (detector)
signal comes later than the SYNC signal (forward start–stop mode). In addition, the delay must be chosen so
that it fits in the measurement range of the histogrammer (max. 67 µs). To obtain such a timing it may be
necessary to adjust the input offsets.
If you have an appropriate setup installed and a measurement running, you can slightly vary all levels
(including SYNC) for best timing response. If there is no clear optimum for any level, return to the center of its
stable working range. For optimization purposes you also may want to switch to a higher resolution. If the
rough setup is complete and a time–correlated signal peak is present in the histogram, the Full Width Half
Maximum (FWHM) of the signal peak is displayed on–line as a figure of merit. You can adjust the input offsets
to place the signal properly within the boundaries of the SYNC frame. You can also adjust your axis limits to
optimize the display. Note that you can double–click all the rate meters to enlarge them as separate windows.
This is useful for optical alignment work, if the PC screen is in some distance from your optical table.
Input Troubleshooting
Whenever there is a problem, first check your cabling, detector power supply and SYNC source. To be
absolutely sure, check the signals with a fast oscilloscope (50 Ω input). Disconnect the scope when you are
done. Never try to deliver the signals to multiple 50 Ω loads in parallel, using simple T–pads. If you have
multiple detectors, try to test your setup with one detector first to keep things simple.
If you cannot get a stable SYNC rate reading of expected figures, there may be several reasons:
● there is no proper SYNC signal (voltage, polarity, pulse width, frequency)
● the SYNC CFD level setting is inappropriate
● the SYNC divider setting is inappropriate
● Note that at small rates the meter display may fluctuate between two discrete values.
Reasons for zero input channel counts are:
● wrong input selection for the rate meters
● no or inappropriate signal (voltage, polarity, pulse width, frequency)
● inappropriate CFD levels
● preamplifier or detector failure
Note that all rate meters are updated at the display refresh rate you have selected in the general settings
dialog. In oscilloscope mode the update frequency is equal to the acquisition time. If nothing seems to happen
at all, you may be in oscilloscope mode with a very long acquisition time set.
Once count rates > 0 are being displayed you should then also see counts appearing in the histogramming rate
meter, if your measurement range is large enough. Make sure the offset is set to 0. If there are histogramming
counts but no histogram is building up on the screen, check the time and count axis bounds. It is best to start
with a wide display range and then narrow it down. Similarly, a logarithmic count axis setting is the safest way
to see even small histograms. If your measurements stop earlier than expected, make sure the Stop at level is
not set to less than 4,294,967,295 unless you have experimental reason to limit the count height.
In the process of set-up you should observe the warning icon that may appear at the bottom right of
the main window. When the HydraHarp software is running with functional hardware it continuously
collects information about the input signals and the current acquisition settings. If these settings
together with the input rates indicate possible errors, the software will activate the warning icon.
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The warning icon can be clicked to display a list of current warnings together with a brief explanation of each
warning (see also section 8.1). Note that the software can detect only a small subset of possible error
conditions. It is therefore not safe to assume “all is right” just by seeing no warning. On the opposite, if any of
the warnings turns out to be an unnecessary nuisance, e.g. because your specific measurement conditions will
expectedly and inevitably cause it, you can disable that warning via the general settings dialog (see section
6.7).
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5.2. Setting Up and Running Interactive Measurements
The primary mode of operation of the HydraHarp software is interactive histogramming mode. This is what the
main window of the software is dedicated to. The user can set up measurement parameters, start
measurements and immediately see histogram data on the screen. In further sections, e.g. on TTTR mode, you
will learn about other modes of operation with less user interaction that will collect data straight to disk without
immediate visualization. Here, we focus on the interactive histogramming mode of operation.
To set up measurement parameters use the HydraHarp control panel. The control panel
can be opened by clicking the control panel button on the toolbar or by pressing <Alt>+C.
In the control panel section 'Acquisition' you can set the resolution (time per
bin), the offset, the measurement time, and the block of memory to use for
this measurement. To begin, use a measurement time of 1 second and an
offset of 0. There are always 65,536 time channels (bins) per HydraHarp
histogram. Histograms can be measured in 512 memory blocks. Out of these
up to 8 curves can be displayed and one ‘active curve’ can be used for a
measurement. You can designate the active memory block you wish to use for
the next measurement by selecting the block number in the control panel.
Never forget to select a new block when collecting new data and old data is
intended to be preserved.
Use the trace mapping dialog to select up to 8 curves for display. Make sure the memory
block you measure into is mapped to a display trace that is switched on, so that you can
see the trace. You can reach the trace mapping dialog from the toolbar or by clicking on
the trace colour indicator next to the block selector.
The colour indicator shows the trace colour the chosen block is currently mapped to. If it
is a solid square, the curve is mapped and shown. If it is mapped but not shown, the
indicator shows a small striped square. If the curve is not even mapped for display the
indicator remains white.
There are two basic histogramming modes for interactive measurements: Oscilloscope
and Integration mode. Oscilloscope mode repeatedly collects histograms with a fixed
measurement time and displays them on the screen. This lets you see fast changes in the
histogram, e.g. for optical setup and adjustments. Usually this only makes sense with
relatively strong signals and short acquisition times. Integration mode is usually operated
with longer acquisition times. In this case the histogram continues to grow over a longer
time and the display is updated at regular intervals, so that the accumulation process can
be observed.
To start a measurement with the current control panel settings, use the start button (GO)
on the toolbar or press <Alt>+G.
To stop a measurement use the stop button on the toolbar or press <Alt>+S.
Note that a measurement may automatically stop and / or restart, dependent on the
current settings of ‘stop on overflow’ and ‘restart’ in the control panel.
Note: to actually run a meaningful measurement you will need to set up the input
channels, most importantly with appropriate CFD levels as outlined in the previous section.
Also allow a warming–up period of 20 to 30 minutes dependent on lab temperature before
using the HydraHarp for final measurements. You can use this time for set–up and
preliminary measurements. Click the calibrate button after warming up.
The rate meters (bottom of main window) permit visual control of the data acquisition. Note the selector at the
right of the rate meters. This selects the channel the rate meters are referring to. Select the channel you are
currently using. The meters are often too small to view from further away, e.g. when adjusting the optical setup.
You can in this case simply double–click the rate meter of interest. This opens a large meter display that you
can then size and move on the screen as you like.
Once you have established standard settings for your experimental setup you may want to save them to a file.
The control panel settings can then be recalled at any time. The program stores all settings together with the
histogram data of those curves in memory (max. 512) that have been filled by a measurement. In addition to
this, all settings are memorized in the Windows registry, so that at program startup you find the control panel as
it was when you last closed the HydraHarp software.
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5.3. Time Tagged Mode Measurements
Time–Tagged Time–Resolved (TTTR) mode allows the recording of individual count events directly to hard
disk without on–board forming of histograms. It originates from the TimeHarp TCSPC systems and has been
further improved for the HydraHarp 400.
In TTTR mode, the timing of individual events is faithfully recorded to disk rather than just histogrammed. This
time-tagging is particularly interesting where the dynamics in a fluorescence process are to be investigated.
The availability of the time–tags permits photon burst identification, which is of great value e.g. for Single
Molecule Detection in a liquid flow. Other typical applications are Fluorescence Correlation Spectroscopy
(FCS) and Burst Integrated Fluorescence Lifetime (BIFL) measurements. Together with an appropriate scan
controller, TTTR mode is also suitable for ultra fast Fluorescence Lifetime Imaging (FLIM).
The HydraHarp 400 actually supports two different Time–Tagging modes, T2 and T3 mode, which will be
explained further below. When referring to both modes together we use the general term TTTR.
5.3.1. System Requirements
In cases where the time–tagging modes are to be used with high continuous count rates (> 1 Mcps) the PC
system must meet some special performance criteria, that usually are insignificant in standard interactive use
of the HydraHarp. The reason for this is the relatively large amount of data being generated in TTTR mode. In
order to prevent an overflow in the recording, the data must be transferred to the computer in real–time. This
requires a modern PC with a fast I ⁄ O subsystem. A recent CPU running at least at 1 GHz is necessary. For
the best possible performance in TTTR mode a modern SATA 6G hard disk with high throughput is
recommended. If it is intended to make use of the full bandwidth of a HydraHarp with USB 3.0 interface then
the hard disk must be able to handle sustained write rates of 160 MBytes/s. This can be achieved with RAID
arrays or modern solid state disks.
At the same time, the USB subsystem must be configured correctly. In order to deliver maximum throughput,
the HydraHarp 400 uses state–of–the–art USB 2.0/3.0 bulk transfers. This is why the HydraHarp must rely on
having a suitable host interface of corresponding speed rating. USB 2.0 host controllers of modern PCs are
usually integrated on the mainboard. USB 3.0 interfaces may need to be upgraded as PCIe cards. Throughput
is usually limited by the host controller and not the HydraHarp. Make sure that the host controller drivers are
correctly installed. This may require using the mainboard manufacturer's driver disk rather than relying on
Windows drivers. Do not run other bandwidth demanding devices on the same USB interface when working
with the HydraHarp.
5.3.2. T2 Mode
In T2 mode all timing inputs of the HydraHarp 400 and the SYNC input are functionally identical. There is no
dedication of the SYNC input channel to a sync signal from a laser. It may be left unconnected or can be used
for an additional detector signal. Usually all regular inputs are used to connect photon detectors. The events
from all channels are recorded independently and treated equally. In each case an event record is generated
that contains information about the channel it came from and the arrival time of the event with respect to the
overall measurement start. The timing is recorded with the highest resolution the hardware supports (currently
1 ps). Each T2 mode event record consists of 32 bits. There are 6 bits for the channel number and 25 bits for
the time–tag. If the time tag overflows, a special overflow record is inserted in the data stream, so that upon
processing of the data stream a theoretically infinite time span can be recovered at full resolution. Dead times
exist only within each channel (80 ns typ.) but not across the channels. Therefore, cross correlations can be
calculated down to zero lag time. This allows powerful new applications such as FCS with lag times from
picoseconds to hours. Autocorrelations can also be calculated at the full resolution but of course only starting
from lag times larger than the dead time.
The 32 bit event records are queued in a FIFO (First In First Out) buffer capable of holding up to 2 M event
records. The FIFO input is fast enough to accept records at the full speed of the time–to–digital converters (up
to 12.5 Mcps each). This means, even during a fast burst no events will be dropped except those lost in the
dead time anyhow. The FIFO output is continuously read by the host PC, thereby making room for fresh
incoming events. Even if the average read rate of the host PC is limited, bursts with much higher rate can be
recorded for some time. Only if the average count rate over a long period of time exceeds the readout speed of
the PC, a FIFO overrun could occur. In case of a FIFO overrun the measurement must be aborted because
data integrity cannot be maintained. However, on a modern and well configured PC a sustained average count
rate of 9 Mcps is possible with USB 2.0. If your device supports USB 3.0 and is connected appropriately the
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throughput can be as high as 40 Mcps. This total transfer rate must be shared by the input channels. For all
practically relevant photon detection applications the effective rate per channel is more than sufficient.
For maximum throughput, T2 mode data streams are normally written directly to disk, without preview other
than count rate and progress display. However, it is also possible to analyze incoming data ”on the fly”. The
HydraHarp software provides a real-time correlator for preview during a T2 mode measurement (see section
5.3.7). Other types of real-time processing must be implemented by custom software. The HydraHarp software
installation CD contains demo programs to show how T2 mode files can be read by custom software. The
implementation of custom measurement programs requires the HydraHarp programming library, which is
available as a separate option.
5.3.3. T3 Mode
In T3 mode the SYNC input is dedicated to a periodic sync signal, typically from a laser. As far as the
experimental setup is concerned, this is similar to histogramming mode. The main objective is to allow high
sync rates (up to 150 MHz) which could not be handled in T2 mode. Accommodating the high sync rates in
T3 mode is achieved as follows: First, the sync divider is employed as in histogramming mode. This reduces
the sync rate so that the channel dead time is no longer a problem. The remaining problem is now that even
with a divider of 16, the sync rate is still too high for collecting all individual sync events like ordinary T2 mode
events. Considering that sync events are not of primary interest, the solution is to record them only if they arrive
in the context of a photon event on any of the input channels. This is the same as the original TTTR mode
concept as it was first conceived for the TimeHarp system. The event record is then composed of two timing
figures: 1) the start–stop timing difference between the photon event and the last sync event, and 2) the arrival
time of the event pair on the overall experiment time scale (the time tag). The latter is obtained by simply
counting sync pulses. From the T3 mode event records it is therefore possible to precisely determine which
sync period a photon event belongs to. Since the sync period is also known precisely, this furthermore allows
to reconstruct the arrival time of the photon with respect to the overall experiment time.
Each T3 mode event record consists of 32 bits. There are 6 bits for the channel number, 15 bits for the start–
stop time and 10 bits for the sync counter. If the counter overflows, a special overflow record is inserted in the
data stream, so that upon processing of the data stream a theoretically infinite time span can be recovered.
The 15 bits for the start–stop time difference cover a time span of 32,768×R where R is the chosen
resolution. At the highest possible resolution of 1 ps this results in a span of 32 ns. If the time difference
between photon and the last sync event is larger, the photon event cannot be recorded. This is the same as in
histogramming mode, where the number of bins is larger but also finite. However, by choosing a suitable sync
rate and a compatible resolution R, it should be possible to reasonably accommodate all relevant experiment
scenarios. R can be chosen in doubling steps between 1 ps and 33.5 µs.
Dead time in T3 mode is the same as in the other modes (80 ns typ.) Within each photon channel,
autocorrelations can be calculated meaningfully only starting from lag times larger than the dead time. Across
channels dead time does not affect the correlation so that meaningful results can be obtained at the chosen
resolution, all the way down down to zero lag time. This requires dedicated software.
The 32 bit event records are queued in a FIFO (First In First Out) buffer capable of holding up to 2 M event
records. The FIFO input is fast enough to accept records at the full speed of the time–to–digital converters (up
to 12.5 Mcps each). This means, even during a fast burst no events will be dropped except those lost in the
dead time anyhow. The FIFO output is continuously read by the host PC, thereby making room for new
incoming events. Even if the average read rate of the host PC is limited, bursts with much higher rate can be
recorded for some time. Only if the average count rate over a long period of time exceeds the readout speed of
the PC, a FIFO overrun could occur. In case of a FIFO overrun the measurement must be aborted because
data integrity cannot be maintained. However, on a modern and well configured PC a sustained average count
rate of 9 Mcps is possible. If your device supports USB 3.0 and is connected appropriately, the throughput can
be as high as 40 Mcps. This total transfer rate must be shared by the input channels. For all practically relevant
photon detection applications it is more than sufficient.
For maximum throughput, T3 mode data streams are normally written directly to disk. However, it is also
possible to analyze incoming data ”on the fly”. One such analysis method is the on–line correlation
implemented in the HydraHarp software. Other specialized analysis methods must be implemented by custom
software. The HydraHarp software installation CD contains demo programs showing how T3 mode files can be
read. The implementation of custom measurement programs requires the HydraHarp programming library,
which is available as a separate option. Another alternative for advanced TTTR data collection and analysis is
the SymPhoTime software offered by PicoQuant.
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5.3.4. Running a basic TTTR Mode Measurement
A TTTR mode measurement (T2 or T3 mode) will typically be started after all control panel settings have been
tested in normal interactive histogramming mode (oscilloscope or integration). The acquisition time
(measurement time) and the file for saving the data are the only parameters that can be set separately.
A typical approach to set up a TTTR mode measurement would be by first starting oscilloscope mode with an
acquisition time of e.g. 1 second. Then all control panel settings should be optimized to reliably obtain the
expected data.
Once all settings are satisfactory, click the “TTTR Mode“ button on the toolbar. This will bring up the
“TTTR Mode“ dialog.
The dialog section Acquisition Settings is for the selection of the measurement mode (T2 / T3), an overall
acquisition time and a file name. Note that switching between T2 and T3 mode takes some time because the
hardware must be reconfigured. Normally such a switching should not occur often because the two modes
usually require a very different experiment setup.
The section Acquisition Settings also has two tick boxes for the handling of existing files. You can turn on a
warning and / or automatically have numbers appended to the file name, so that you can conveniently perform
series of measurements. The file name is shown in red if the file already exists. The button with the file icon will
open a standard Windows file dialog. You can select an existing file or choose a new name. The HydraHarp
TTTR mode files (starting from software version 3.0) have the file name extension .ptu. For maximum count
rate throughput you should choose a file destination on a fast local hard disk as outlined above. Network drives
are often too slow.
The dialog section Status shows elapsed time, the count rates and the number of collected records. Below
these figures there is a status line showing what is currently happening. Further below there are buttons for
Start, Stop and Cancel. Start and Stop control the actual TTTR measurement run. Cancel is for leaving the
TTTR mode dialog.
The TTTR mode measurement will start as soon as you click the Start button. You will then be able to watch
the progress of your measurement in the status boxes. The leftmost status box shows the elapsed
measurement time. The two boxes in the center show the count rates. The rightmost box will show the number
of events that have been recorded. Note that this includes overflow and marker records. Therefore, the number
of records will be somewhat larger than the true photon counts. The overflow and marker records will be
removed by the data analysis / processing software.
Next to the status line you may see a warning icon. When the TTTR mode dialog is running it
continuously collects information about the input signals and the current acquisition settings. If these
settings together with the input rates indicate possible errors, the software will activate the warning
icon. The warning icon can be clicked to display a list of current warnings together with a brief
explanation of each warning (see also section 8.1). In order to fix wrong settings you may have to
close the TTTR mode dialog and return to the control panel.
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A measurement can be stopped at any time by clicking the Stop button. The data recorded up to this point will
be in the file. When the measurement has completed, the Stop button will change to grey (disabled). Use the
Cancel button to return to the normal interactive mode. Again, this will take some time for hardware
reconfiguration.
5.3.5. External Markers
Often it is desirable to synchronize TCSPC measurements with other information or processes of complex
measurement tasks. In order to perform e.g. Fluorescence Lifetime Imaging, the spatial origin of the photons
must be recorded as well. For this purpose one needs a mechanism to assign external synchronization
information to the TCSPC data. For the special case of Fluorescence Lifetime Imaging, conventional systems
use large arrays of on–board memory and switch to new blocks of memory upon arrival of e.g. a pixel clock
pulse. To accommodate the large amount of data generated due to the 3–dimensional matrix of pixel co–
ordinates and lifetime histogram channels causes serious problems. Even with modern memory chips, this
approach is limited in image size and / or number. In addition, it is expensive, and implies loss of information
about the individual photon arrival times. To solve the problem much more elegantly, the TTTR data stream
generated by the HydraHarp can contain markers for synchronization information derived from an imaging
device, e.g. a scan controller. For this purpose the front panel of the HydraHarp SYNC module HSR 110
provides four TTL inputs for synchronization signals (M1..M4).
The figure below illustrates how the external marker signals are recorded in the data stream.
Bullets denote a photon, blue pulses denote a marker signal. The external markers are treated almost as if they
were regular photon event records. A special channel code allows to distinguish true photon records from the
marker records. Software reading the TTTR file can thereby filter out the markers, e.g. for line and frame clock
in imaging applications. This makes it possible to reconstruct the 2D or 3D image from the stream of TTTR
records, since the relevant XYZ position of e.g. a scanner can be determined during the data analysis. The
data generated is nearly free of redundancy and can therefore be transfered in real–time. The image size is
unlimited both in XYZ and in count depth. Since there are up to four such synchronisation signals, all imaging
applications can be implemented and even other experiment control signals can be recorded. This marker
scheme is a very special innovative feature of the PicoQuant TCSPC electronics. It is worth noting that this
technology enabled PicoQuant GmbH to develop the leading edge MicroTime 200 Fluorescence Lifetime
Microscope.
The TTL compatible inputs accept the synchronization signals that will be recorded as markers. The active
edges of these signals can be chosen in the general settings dialog (available through the Toolbar). Both high
and low state must be at least 50 ns long. The period may therefore (in principle) be as short as 100 ns but
data bus throughput constraints will apply. Each marker creates an additional TTTR record, so that one must
ensure not to swamp the data stream with too many marker records. Markers also take precedence over
photon records, so that excess marker traffic can suppress photon records. In fast imaging applications it is
therefore recommended not to use a pixel clock but a line clock only. Since each photon has a time tag, it is
usually not necessary to use an additional pixel clock. The accuracy of marker timing is on the order of 50 ns.
For each channel there is another TTL input that can be used to externally enable / disable the recording of
incoming counts (see section 8.3.2). Counting is enabled when this input is held high (or left open). Pulling it
low disables counting. This mechanism can be used to blank out periods where e.g. an external scanner is
moving to the next position. The count enable signal is also active in interactive mode. It may be used to
synchronize the start of acquisition with external events.
5.3.6. Using TTTR Mode Data Files
For diagnostic purposes you may re–load a T3 mode file into the HydraHarp software. The limitation is that you
will only be able to form a histogram over the start–stop times in your T3 mode data. The time–tag information
will not be used here. The HydraHarp software will recognize that you are loading a T3 mode file and how
many records there are contained in it. It will then prompt you for a range to use for histogramming. The
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histogram will go to blocks 0..N-1 of the histogram memory where N is the number of channels that were used
in the T3 mode measurement. A TTTR mode file also contains all control panel settings that were active at the
time of the measurement run. After loading a TTTR mode file, you will find the document title reading
“Histogram from...”. If you choose to save such data you will have to give it a new file name ( *.phu). This is
because now a histogram has been formed, and saving it to the same file name would destroy the original
TTTR mode file. However, you may save the previously formed histogram as if it were obtained in normal
interactive mode, to a standard HydraHarp histogram data file (*.phu).
Reloading T3 mode files serves as a quick diagnostic tool only. For T2 mode files such a feature is currently
not available. Further processing or analysis of TTTR mode data must therefore be performed through external
data analysis software. Such software is available from PicoQuant for a wide range of analysis tasks
(SymPhoTime). Further specialized analysis can be performed by dedicated custom software. If you wish to
save the cost for the commercial TTTR analysis software or if you require special analysis algorithms you may
want to program your own analysis software. For development of your own custom programs, please refer to
the demo code provided. Demo source code is contained on your HydraHarp installation disks and will be
installed by the software setup. Also see section 8.2 for the file format specifications. The paragraph below can
only give an outline.
The first part of a TTTR mode file is a header with the basic setup information, similar to that of the other
modes. What follows after the header is a sequence of 32 bit TTTR records. The TTTR records in the file
consist of different pieces of information in groups of bits. These pieces of information must be extracted by a
bit masking / shifting operation. Their specific layout is different for T2 and T3 mode. In both cases, in addition
to extracting the bit fileds, the software must step through the whole file and interpret the overflow records and
correct the overall time axis accordingly. Details on how this can be done should be looked up in the demo
programs that are installed as part of the software distribution.
Note that T2 and T3 mode files cannot be distinguished by their file extension. They all get stored as *.ptu .
However, you can use the PicoQuant File Info shell extension that will be installed by the HydraHarp software
setup to inspect individual header items of a *.ptu or *.phu file. This includes the measurement mode. Just
right-click on the file in Windows explorer and select Properties. Then look at the tab PQ File Info.
5.3.7. TTTR Mode Measurements with Real–Time Correlation
An important application of the HydraHarp and its TTTR mode is Fluorescence Correlation Spectroscopy.
While TTTR data collection and off–line software correlation has been used for quite some time, PCs have only
recently become fast enough to calculate the correlation function “on the fly” while the data is being collected.
This capability can be very useful in setting up and monitoring of FCS experiments. The HydraHarp software
provides such a real–time software correlator for T2 and T3 mode.
Although it collects the same TTTR mode files, the real–time correlator has its own button on
the Toolbar.
The next figure shows the TTTR mode correlator dialog. Its lower section provides the same file handling and
progress indicators elements as in plain TTTR mode. Please refer to section 5.4 for details. The upper section
of the dialog contains the correlator display and various control elements.
The dialog section Cor(Ax,Bx) allows to select individual input channels for the correlation. A and B are the
virtual input channels to be correlated. All input channels that are ticked in columns A resp. B will be used in
the corresponding correlation channel. If your selection is empty, the heading of the section Cor(Ax,Bx) will turn
red to indicate the problem. If you proceed anyway you will get error messages upon start.
The correlator always calculates the autocorrelations of the two virtual channels (AA and BB) as well as the
cross–correlation (AB). There are three tick boxes that select which of these are shown.
The dialog section Accumul. allows to select an update time for the correlation display. Furthermore it allows to
select between repetitive updates (Osc.) and cumulative collection (Int.). If the latter is chosen one can
manually reset the correlator by clicking the Reset button. Note that all these settings affect only the real–time
correlator. The raw TTTR mode data will be collected continuously and completely independent from the
correlator settings.
The Save button under the Accumul. section allows to save the correlator results as they were last shown. The
saved result is an ASCII file with some header information. The format is self–explanatory. Note that the real–
time correlator is primarily a tool for preview during measurement setup and optimization. It does not allow to
correlate data from TTTR files. If you need the correlation over the entire stream of collected data you need to
make sure you run in Integrating mode and do not press reset during the measurement. If you need the
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correlation results you should save them here. If you need to perform further or more thorough FCS analysis on
the collected data you can use the SymPhoTime software from PicoQuant.
On the right hand side there are controls for the axis ranges of the correlator display. Note that they affect only
the display. Collected data is always complete, independent from the axis settings. In contrast, the selection
box subsmpl has an effect on the correlator results. It determines how many tau points are calculated. The
correlator works in a logarithmic multiple tau scheme and subsmpl specifies the number of linear subsamples
in each log stage. A higher number of subsamples increases the resolution of the correlation curve. Calculating
more points is more time consuming and therefore it may lead to lower count rate limits that can be handled. In
this case you may get FIFO overruns. The default of subsmpl=8 is a reasonable trade–off between speed and
resolution. On faster computers the setting of subsmpl=16 is a good choice.
Note that the starting point and spacing of the Tau sampling points also depends on the time tag resolution. In
T3 mode this corresponds to the sync period. In T2 mode the native resolution of 1 ps is binned down to a time
tag resolution of 25 ns in order to make the data manageable for the real-time correlation algorithm.
The correlation curve display can be shown with a grid (see general settings dialog, accessible from the toolbar
of the main window). It also shows two useful figures obtained from the collected data (top right of curve
window). The first figure is an approximation of G(0). In classic FCS experiments this corresponds to the
inverse of the number of particles in the focal volume. It is continuously updated together with the curve
display, which can be useful for system ajdustment, notably when using the repetitive accumulation mode
(Osc.). Note that the approximation is a simple averaging over the first ten Tau points. The figure B is an
indicator for molecular brightness. It is also updated continuously. Note that it is also only an approximation
obtained by multiplying the G(0) approximation with the average count rate on both virtual correlation channels
A and B. Dependent on the chosen channels in A and B this may lead to figures that are not truly molecular
brightnesses, however, as an adjustment aid they should be useful in any case.
All other aspects of TTTR data collection with correlator preview are the same as in plain TTTR mode as
described in the previous subsections. Topics such as using external markers or how to use the data files
should be looked up there.
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5.4. Time–Resolved Excitation and Emission Spectra
In time–resolved fluorescence research it is often of great interest to observe time–dependent spectral shifts
and decay changes, e.g. in the context of solvent relaxation dynamics and general spectral evolution. This
requires recording Time–Resolved Excitation/Emission Spectra (TRES), ideally with automated wavelength
scan and TCSPC data collection under full software control.
In order to measure excitation or emission spectra in combination with fluorescence lifetime, the HydraHarp
software provides an automated TRES measurement mode. This mode allows to control a monochromator via
a stepper motor and automated collection of spectrally resolved lifetime histograms. Data is collected as in
standard “Integration Mode” and saved in different blocks of memory for each wavelength. Note, however, that
TRES data is always collected through input channel 1 only.
If a monochromator with appropriate stepper motor and associated drivers is installed, a specialized dynamic link library (Mono.dll) can be installed together with the HydraHarp software. The
presence of this DLL will be detected by the software. In this case the button for Monochromator
and TRES control on the toolbar will be enabled.
Note that most of the monochromators can be controlled only through dedicated stepper motor hardware from
PicoQuant or selected vendors, usually installed as part of a complete PicoQuant spectrometer. Custom confi gurations can only be supported upon special request.
Clicking the monochromator button will launch a dialog for manual monochromator control and TRES setup.
There you can set up parameters such as the start, step and end of the wavelength scan. There will be error
messages if the monochromator / stepper and associated drivers are not configured properly.
The monochromator used for TRES measurements is controlled by a special dialog. It shows a tab control and
a status panel at the bottom of the dialog. The three tabs give access to the Initialization, TRES and Manual
page of the dialog.
The status panel shows the current monochromator position in nm and whether the monochromator is currently
moving (the red "LED" labelled Stepping is flashing, when it moves).
The dialog is "non modal", i.e. it does not need to be closed before other windows can be accessed. It has its
own icon in the Windows Taskbar, from where it can be brought back to the top, should it have been covered
by other windows.
Monochromator Models
The type of the current monochromator is displayed in the title bar of the dialog. Apart from a simulation mode,
as of this release the following monochromator models are supported: Sciencetech ST–9030, ST–9030DS and
ST–9055, Acton Research SP–2150, SP–2155, SP–150 and SP–275. Some other Acton Research models are
partially supported. Note that some monochromators can only be controlled through dedicated stepper motor
hardware from PicoQuant or the monochromator supplier. Usually these are installed as part of a complete
PicoQuant spectrometer. Please contact PicoQuant for details.
If your system is not configured for use of a real monochromator, the Monochromator / TRES dialog shows the
caption Simulation. This is for demonstration purposes only. It will work the same way as the "real" ST–9030
monochromator described below, except that it controls no real hardware and just simulates the
monochromator adjustments. Of course the simulated monochromator adjustments will not have any effect on
the measured TCSPC histograms.
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Monochromator Initialization
Before use, the monochromator must be initialized. This means, it has to seek its mechanical reference
position (determined by a micro switch). Physical calibration values are kept in a monochromator configuration
file (monochromator.cfg) which is generated by the system supplier at the time of delivery. Do not edit this file
unless you have exact instructions for doing so.
Position initialization is required each time the software is started. It can be controlled on the Initialization page
of the dialog. At first startup the dialog shows the state "not initialized". This means the software has no infor mation about the current monochromator / stepper position.
The position initialization is started by the Initialize button. If the initialization is running, the caption of the
button will change to Stop and the LED will start flashing. You should also hear the stepper motor. If the
initialization is successful, the string "not initialized" will change to "Initialization OK", otherwise it will change to
"Initialization failed!". In this case, check cabling and power supply of the system. This may happen if either the
Stop button was pressed or an error occurred during the initialization process. The Manual and TRES pages
will not be available unless the initialization was successfully performed. If for any reason the initialization
becomes invalid (e.g. if the monochromator was turned manually and subsequently accidentally reached the
end point switch) the Initialization page will be activated and access to the Manual and TRES pages will be
blocked.
Manual Monochromator Control
The Manual page of the dialog can be used to position the monochromator manually at a given wavelength.
The desired position can be entered in the Position edit box. Clicking Apply or Pressing <Enter> will cause the
monochromator to move to the specified position. Alternatively, the spin buttons right to the edit box can be
used to modify the position. The step width of this action is defined in the D (delta) edit box. Pressing arrow
buttons will make the change take effect immediately; entering values by keyboard require pressing Apply or
<Enter> to show an effect.
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If there is a large difference between the current monochromator position and the position entered, the
movement will take some time. If you wish to interrupt this process, press the Stop button. The monochromator
immediately will slow down and stop. The deceleration is necessary to preserve the calibration.
Running TRES Measurements
The TRES page of the dialog contains the controls used to set up an automated scanning of the
monochromator. The start and stop wavelengths of the scan can be entered in the corresponding edit boxes,
the D (delta) box defines the step width of the scan. The scan starts always exactly at the Start value. If the
Stop position cannot be reached by an integer multiple of the step width, the scan stops at the last position
within the start / stop range. If the start position is larger than the stop position, the scanning direction will be
reversed. The maximum range and number of wavelength steps is determined by the physical characteristics
of the monochromator as well as the maximum number of memory blocks to record histograms in (512). Again,
pressing the spin buttons will apply the change in the corresponding edit boxes immediately; entering values
manually require pressing <Enter> or the Apply button.
The IRF can be measured automatically as part of the scan, at a separately chosen wavelength. To do so,
check the box entitled IRF at and enter the wavelength at which to measure the IRF in the corresponding edit
box. The monochromator will go to the IRF position at the beginning of the TRES measurement. After the IRF
was measured, the actual TRES scan will be performed. The IRF will always be recorded in memory block 0,
while the wavelength scan always starts in block 1. Note that starting a TRES measurement will clear and
subsequently overwrite all curves in memory that may have been recorded previously. If you wish to measure
the IRF yourself you must do this AFTER the TRES measurement. If the curves in memory were loaded from a
file, the actual file on disk will not be overwritten until you save to the file ( Save menu or corresponding toolbar
button). Use Save As and select a new file name if you want to preserve the old file. Note that a TRES file will
remain a TRES file for its lifetime. This means that after creating or re-loading such a file you can do only two
things: perform a new TRES measurment (effectively overwriting the old file contents) or measure an IRF into
curve 0 (overwiting only curve 0). If you want to perform any other measurement you will need to create a new
file or load a non-TRES file.
The TRES measurement is selected in the HydraHarp control panel. You need to use the selection box for the
measurement mode to select TRES mode (instead of OSC or INT).
A TRES measurement is started or canceled as usual via the toolbar buttons for start
and stop. The data acquisition will be performed like a standard “Integration Mode”
measurement, while the block number is incremented for each new wavelength.
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At the beginning of each TRES run, the active curve (block) will be set to 0, or to 1 if no IRF is collected. All
curves that were previously collected and not saved to a file will be overwritten with new data. During a TRES
run, the currently collected data will always be shown as Trace 0 (dark blue), even though the block (Trace)
number is actually incremented at each wavelength step. This is to overcome the limitation of only 8 display
curves being available. You can watch the block number in the control panel to see the curve currently being
collected. Manual entry will be disabled during the run. Also, you can watch the status bar to see the current
wavelength and stepping activity. Note that for a TRES measurement the control panel setting 'Stop Of' applies
as in Integration mode, while 'Restart' is meaningless in TRES mode.
TRES Data Analysis and Visualization
Having collected a complete TRES data set you can save it to a regular *.phu file. You can also inspect
individual curves via the Trace Mapping dialog. You can furthermore use the software tool FluoPlot from
PicoQuant to visualize and analyze the data in various 2D and 3D representations with a multitude of options
for colouring, scaling and changing view aspects in 3D. The figures below show FluoPlot visualizations from a
TRES measurement of mixed oxazine dyes in ethanol.
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FluoPlot is provided on your software installation CD. Just run FluoPlotSetup.exe from there. If you received
your HydraHarp software by download or email you may need to request FluoPlot separately. Note that the
program requires modern hardware acceleration with support for OpenGL version 1.5 or higher. Speedy
handling of large data files requires sufficient memory. Further data analysis may need to be performed by
dedicated software, either custom programs or specialized solutions available from PicoQuant.
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6. Controls and Commands Reference
6.1. Main Window
The Title Bar
The title bar is located along the top of a window. To move the window, drag the title bar. Note: You can also
move dialog boxes by dragging their title bars. The title bar may contain the following elements:
System Menu
HydraHarp – [Name of the file or dialog]
Minimize Button
Maximize or Restore Button, resp. /
Close Button
Scroll bars
Displayed at the right and bottom edges of the HydraHarp window if a certain minimum window size is
reached. The scroll boxes inside the scroll bars indicate your vertical and horizontal location in the display area.
You can use the mouse to scroll to other parts of the window.
Size command
Use this command to resize the active window.
Note: The command is unavailable for already maximized or minimized windows.
Shortcut
Mouse: Drag the corners or edges of the window.
Keys: <Alt>+<Space> S
Minimize command
Use this command to reduce the HydraHarp window to an icon. Running measurements will continue.
Note: The command is unavailable for already minimized windows.
Shortcut
Mouse: Click the minimize button on the title bar.
Keys: <Alt>+<Space> N
Maximize command
Use this command to enlarge the active window to fill the available space.
Note: The command is unavailable for already maximized windows.
Shortcut
Mouse: Click the maximize button on the title bar;
or double–click the title bar of a not–enlarged window.
Keys: <Alt>+<Space> X
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Restore command
Use this command to return from an enlarged window to the previous size.
Note: The command is only available for already maximized or minimized windows.
Shortcut
Mouse: Click the maximize button on the title bar;
or double–click the title bar of the enlarged window.
Keys: <Alt>+<Space> R
Close command
Use this command to close the active window or dialog box.
Double–clicking a system menu box is the same as choosing the Close command.
Shortcuts
Mouse: Click the close button on the title bar.
Keys: <Alt>+<F4>
<Alt>+<Space> C
Panel Meters
At the bottom of the main window there is a set of panel meters. The meters showing units of cps (counts per
second) in their title are rate meters. The leftmost rate meter shows the Sync input rate. The next meter shows
the channel input rate. Note the selector at the right of the panel meters. This selects the channel the rate
meters (except Sync) are referring to. The other meters show the histogramming rate, the total count in the
histogram, the maximum (peak) count and the position of the maximum. They all depend on the input selector.
Note that the Input selector also has a selection option Sum. In this case the meters are fed from the sum of all
input channels. Also note that the internal rate counters have a gate time of 100 ms. Very low rates can
therefore not be measured very accurately and the meter reading may fluctuate between two values.
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6.2. Menus
6.2.1. File Menu
The File menu offers the following commands:
New Clears all histogram data and restores default settings.
Open Opens an existing histogram file.
Save Saves a histogram file.
Save As… Saves an opened histogram file to a specified file name.
Print Prints the currently displayed histogram.
Page Setup Allows modifying the page layout for printing.
Print Preview Displays the layout as it would appear printed.
Print Setup Selects a printer and printer connection.
1…4 <Recent Filename> Opens one of the four last recently opened files
Exit Exits HydraHarp software.
New command
Use this command to create a blank histogram with the last default settings. You can open an existing
histogram file with the Open command.
Shortcuts
Toolbar:
Keys: <Ctrl>+N
<Alt>+F N
Open command
Use this command to open an existing histogram file. All control panel settings will be restored also. You can
also open HydraHarp histogram files (*.phu) by double clicking them or dragging them onto the HydraHarp
icon. You can revert to a blank histogram and default control panel settings with the New command. You can
also select other file types for loading as histogram data, notably T3 mode files. Loading such files as
histogram data is for diagnostics only and cannot use their full information content.
Shortcuts
Toolbar:
Keys: <Ctrl>+O
<Alt>+F O
File Open dialog box
The following options allow you to specify which file to open:
Look in: Select (i.e. browse into) the directory in which the file that you want to open resides.
Main box In this box you see the content of the chosen directory, filtered by Files of type.
File name: Type or select the filename you want to open. This box lists files with the extension you
select in the Files of type box.
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Files of type Select the type of file you want to open. The default HydraHarp extension is .phu
Save command
Use this command to save the current histogram data to its current name and directory. When you save a
histogram for the first time, HydraHarp displays the Save As… dialog box so you can name your file. This
command is unavailable if a measurement is running. If you want to change the name and directory of an
existing file before you save it, choose the Save As command.
Shortcuts
Toolbar:
Keys: <Ctrl>+S
<Alt>+F S
Save As… command
Use this command to (re–)name the current histogram data and save. The software displays the Save As…
dialog box so you can name your file. To save a file with its existing name and directory, use the Save
command.
File Save As… dialog box
The following fields allow you to specify the name and location of the file you are about to save:
Save in: Select (i.e. browse into) the directory where you want to store the file.
Main box In this box you see the content of the chosen directory, filtered by Save as type.
File name: Type a new filename to save a histogram with a different name. The software
automatically adds the extension you specify in the Save as type: box.
Save as type: Selects a filter on the directory chosen in the Save in: box. Only files that pass the filter
are shown in the dialogs main box. Additionally the software adds the extension
associated with the chosen filter to a given file name if it was given without extension.
Print command
Use this command to print the currently displayed histogram curves. This command presents a Print dialog
box, where you may specify the number of copies, the destination printer, the paper orientation and other
printer setup options.
Shortcuts
Toolbar:
Keys: <Ctrl>+P
<Alt>+F P
Page Setup command
Use this command to change the print layout. This command presents the page setup dialog box, where you
may select or deselect various items to appear in the print. You can use print preview to check the resulting
layout.
Shortcut
Keys: <Alt>+F U
Print Preview command
Use this command to display the layout as it would appear when printed. The main window will be replaced
with a print preview window in which one or two pages will be displayed in their printed format. The print
preview toolbar offers you options to view either one or two pages at a time; move back and forth through the
document; zoom in and out of pages; and initiate a print job.
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Shortcut
Keys: <Alt>+F V
Print Setup command
Use this command to select a printer and a printer connection. This command presents a Print Setup dialog
box, where you specify the printer and its connection.
Shortcut
Keys: <Alt>+F R
1, 2, 3, 4 command (most recently used files)
Use the numbers and filenames listed at the bottom of the File menu to open the last four files you closed.
Choose the number that corresponds with the file you want to open.
Shortcuts
Keys: <Alt>+F 1
<Alt>+F 2
<Alt>+F 3
<Alt>+F 4
Exit command
Use this command to end your HydraHarp session. Save your data before exiting.
Shortcuts
Mouse: Click the close button on the title bar.
Keys: <Alt>+<F4>
<Alt>+F X
6.2.2. Edit Menu
The Edit menu offers only one command:
Copy Copies the displayed curves.
Copy command
Copies the currently displayed curves in ASCII format to the clipboard. This can be used to export histogram
data to spreadsheet or data analysis software, e.g. the FluoFit Fluorescence Decay Fit Software. Note that only
the currently selected curves within the current display limits are copied. If the curves in the display are of
different resolution then all 65,536 channels will be copied. Copying data to the clipboard replaces the contents
previously stored there.
The exported data is plain <Tab> separated ASCII and can be read by most of the common spreadsheet
programs or data analysis software.
This command is unavailable while a measurement is running.
Shortcuts
Keys: <Ctrl>+C
<Alt>+E C
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6.2.3. View Menu
The View menu offers the following commands:
Toolbar Shows or hides the toolbar.
Status Bar Shows or hides the status bar.
Axis Panel Shows the axis settings panel.
Control Panel Shows the control panel.
Trace Mapping Shows the trace mapping dialog.
Toolbar command
Use this command to display and hide the Toolbar, which includes buttons for the most common commands,
such as File Open. A check mark appears next to the menu item when the Toolbar is displayed.
Shortcut
Keys: <Alt>+V T
Status Bar command
Use this command to display and hide the status bar, which describes the action to be executed by the
selected menu item or depressed toolbar button, the current measurement activity and keyboard latch state. A
check mark appears next to the menu item when the status bar is displayed. The status bar is displayed at the
bottom of the HydraHarp main window.
Shortcut
Keys: <Alt>+V S
Axis Panel command
Use this command to display the axis settings panel. The result is the same as the axis panel button in the
toolbar or double clicking the on the histogram axes. The axis settings panel can be closed by clicking its close
button.
Shortcut
Keys: <Alt>+V A
Control Panel command
Use this command to display the control panel. This is the same as the control panel button in the toolbar. The
control panel can be closed by clicking its close button.
Shortcut
Keys: <Alt>+V C
Trace Mapping command
Use this command to display the trace mapping dialog. This is the same as the corresponding button in the
toolbar. The trace mapping dialog can be closed by clicking its close button.
Shortcut
Keys: <Alt>+V M
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6.2.4. Help Menu
The Help menu offers the following commands, which provide you assistance with this application:
Help Topics Offers you the contents list of topics on which you can get help.
Help Index Offers you an index to topics on which you can get help.
Help Search Offers you a means of full text search on all help topics.
Activate Context Help Switches the cursor mode to “point to item” for context help.
About HydraHarp… Displays version information of the HydraHarp software.
Note: Online help (context help) on most functions, dialogs, control items etc. is available via the F1 key.
Help Topics command
This command opens a browser offering a tree view like list of contents, and a help page. Browse through the
contents with your mouse or the cursor keys. <Enter> opens / closes chapters, <Cur Up> / <Cur Down>
turns the pages. The help page to the right of the browser will always be held up–to–date.
Shortcuts
Keys: <F1> (if online help was last recently opened in topics mode)
<Alt>+H T
Help Index command
This command opens a browser offering a index list of keywords and a help page. Browse through the contents
with your mouse or the cursor keys. <Enter> opens the current item, <Cur Up> / <Cur Down> navigates
through the index.
Shortcuts
Keys: <F1> (if online help was last recently opened in index mode)
<Alt>+H I
Help Search command
This command opens a browser offering full text search on words or word groups over the whole online help
volume. You can enter more specific search pattern, using the operators "AND", "OR", "NEAR" and "NOT".
Shortcuts
Keys: <F1> (if online help was last recently opened in search mode)
<Alt>+H S
Activate Context Help
Changes the mouse cursor mode to context help mode. You can choose context sensitive help for a visual
element in the GUI by simply clicking on it.
Shortcuts
Keys: <Shift>+<F1>
<Alt>+H C
About HydraHarp... command
Use this command to display the copyright notice and version number of your HydraHarp software and
hardware, if installed. It also provides access to the PicoQuant Web site and software updates.
Shortcut
Keys: <Alt>+H A
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6.3. Toolbar
Toolbar buttons of special interest are explained in some more detail here:
Context Help
When you choose the Context Help button, the mouse pointer will change to an arrow and question mark. Then
click somewhere in the HydraHarp window, such as on another toolbar button. The help topic will be shown for
the item you clicked.
Axis Panel
Clicking this button opens the Axis Panel. This panel provides controls for the axis limits and lin / log display
switching. Double–clicking the axes opens the same dialog. The dialog is non–modal, so it can remain
permanently open. The panel will remember its last position when closed. When it is opened the next time it will
be at that screen position again.
Data Cursor
Opens the data cursor dialog box. You can use this dialog to mark and retrieve the data values of individual
histogram channels. A pair of cross hairs will be provided for marking data points with the mouse or keyboard.
When the data cursor dialog is activated, the cross hairs will appear in the histogram display area. By clicking
on or near a data point, the current cross hair (black) will snap to that point. At the same time a second cross
hair (grey) will jump to the previously marked point. The data cursor dialog then shows the current count and
time values for these points as well as the corresponding differences (deltas). The data cursor always applies
to one "active" curve only. You can select this active curve at the top of the dialog where it is shown with its
corresponding curve colour. While normally the grey cross hair jumps to the previous data point, you can
modify this behaviour by holding the SHIFT key down while clicking on curve points. The grey cross hair will
then remain at its previous position. This allows finding a particular point while keeping the other one fixed.
Another advanced mode of operation of the data cursor is possible via the LEFT and RIGHT arrows of the
keyboard. Pressing these keys will direct the black cursor to the next left or right point in the curve. Again, the
behaviour of the marker for the previous point (grey cross) can be controlled via the SHIFT key.
Calibrate
Calibrates the time measurement circuits. This is necessary whenever temperature changes > 5 K occurred.
Calibrate after warming up of the instrument before starting serious measurements. Warming up takes about
20 to 30 minutes dependent on the lab temperature. It is completed when the cooling fan starts to run for the
first time. Calibration takes only a few seconds. During calibration the status bar shows the message
CALIBRATING. Calibration is only important for serious measurements. You can use the instrument during the
warming–up period for set–up and preliminary measurements. For very long measurements, allow some more
time for thermal stabilization, calibrate immediately before the measurement commences and try to maintain a
stable room temperature during the measurement. The permissible ambient temperature is 15°C to 35 °C. Do
not obstruct the cooling fan at the back and the air inlets at the bottom of the housing.
Control Panel
Launches the HydraHarp Control Panel. If the control panel is already open, subsequent clicking of this button
will just make the control panel the active window. The dialog is non–modal, so it can remain permanently
open. The panel will remember its last position when closed. When it is opened the next time it will be at that
screen position again, even in the next HydraHarp session.
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Trace Map
Launches the Trace Mapping dialog. The HydraHarp can measure histograms in up to 512 memory blocks. Out
of these, up to 8 curves can be displayed. The trace map dialog is used to select the curves to display. Tick the
individual "Show" boxes to display a curve. Select the number of the memory block you wish to map the
individual display curve to. Choose the active memory block you wish to use for the next measurement in the
HydraHarp control panel.
TTTR Mode
Opens the TTTR mode dialog box. Use this dialog to enter the acquisition time and the destination file for a
TTTR mode run. Make sure all other measurement parameters have been set and tested in interactive mode
before entering TTTR mode.
TTTR Real–Time Correlator
Opens the TTTR mode real–time correlator dialog box. Use this dialog for FCS preview during a TTTR mode
run. Make sure all other measurement parameters have been set and tested in interactive mode before
entering TTTR correlator mode. Changing to this mode takes a few seconds for reconfiguration of the
hardware. See also section 5.4.
Monochromator / TRES
In order to measure Time–Resolved Emission Spectra (TRES), the HydraHarp software provides a TRES
measurement mode, that allows to control a monochromator via stepper motors and automated collection of
spectrally resolved lifetime histograms. Clicking this button will launch the dialog for manual monochromator
control and TRES setup. There you can set up parameters such as the start, step and end of the wavelength
scan. There will be appropriate error messages if the monochromator / stepper and associated drivers are not
configured properly.
General Settings
Opens the software settings dialog box. Use this dialog to change standard settings of the HydraHarp software.
Notably these are: Display rate (0.1 to 1s), Draw mode (lines, stairs), Grid–checkbox, Prompt overwrite
(warning before overwriting existing data) and TTTR Marker Settings. The control connector of the HydraHarp
400 provides TTL inputs for synchronization signals. The markers can be enabled resp. disabled and recorded
at the rising or falling edge of the corresponding TTL signal. The active edges can be chosen here. The dialog
also allows selective disabling of warnings that you do not wish to receive. All settings will be kept in the
Windows registry and will be retrieved at the next program start. They will be individual to each user.
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6.4. Control Panel
The control panel consists of several pages (tabbed sheets) containing edit boxes and other controls for related
parameters. These pages are described in the following subsections. Note that the settings of the control panel
as well as the position of the control panel on the screen will be stored in the registry and retrieved at the next
program start. When you load a HydraHarp data file the settings of the control panel will change according to
the settings in that file. This can be used as a means of reverting to a standard setting for the given experiment.
6.4.1. Sync–Input
The settings in this group configure the behaviour of the Sync input. See section 5.1 “Setting Up the Input
Channels“ to get started.
Zero Cross edit box and spin control
Here the zero cross level of the Constant Fraction Discriminator (CFD) for the Sync input can be set. Units are
millivolts (mV), the permitted range is 0 to 40. Type the value as an integer in the edit box and press <Enter>
or click on Apply. Alternatively, use the spin control (next to it) to increment / decrement the current value. In
this case changes take effect immediately without pressing <Enter>.
Discriminator Level edit box and spin control
Here the discriminator level of the Constant Fraction Discriminator (CFD) for the Sync input can be set. Units
are millivolts (mV), the permitted range is 0 to 1,000. This corresponds to real input voltage levels of
0 to −1,000 mV. Type the value as an integer in the edit box and press <Enter> or click on Apply.
Alternatively, use the spin control (next to it) to increment / decrement the current value. In this case changes
take effect immediately without pressing <Enter>.
Offset edit box and spin control
Shifts the relative timing of Sync and photon events and is designed to compensate optical and electrical
delays (e.g. in cables and optical paths), e.g. in order to shift your fluorescence decay within the sync frame so
that it is not truncated. This feature completely eliminates the need for adjustable delay boxes.
Units are picoseconds (ps), the permitted range is from −99,999 ps to +99,999 ps. Type the value as an integer
in the edit box and press <Enter> or click on Apply. The offset value can be entered in steps of picoseconds.
Alternatively, use the spin control next to the edit box to increment ⁄ decrement the current value. In this case
changes take effect immediately without pressing <Enter>.
Sync Divider edit box and spin control
Here the programmable divider of the Sync input can be set. This allows to reduce the sync input rate so that
the period is at least as long as the dead time. This is required for fast sync sources (>12.5 MHz) Internal logic
determines the sync period and re–calculates the sync signals that were divided out. It must be noted that this
only works with stable sync sources that provide a constant pulse–to–pulse period, but all fast laser sources
known today meet this requirement within an error band of a few picoseconds. With random and low rate
signals (< 1 MHz), the divider must be set to 'None'.
6.4.2. Inputs 1…4
The settings in this group configure the behaviour of the input channels 1..4. See section 5.1 “Setting Up the
Input Channels“ to get started. The HydraHarp device is modular and can have up to 8 input channels. In order
to avoid excessive space requirements as well as unpleasant clutter in the controls for all those channnels,
they are organized in groups of 4. This page holds the controls for the input channels 1..4. If only two channels
are installed, only the control elements for channels 1..2 will be accessible. If more than 4 channels are
installed, the control elements of channels 5..8 will be located in a separate page of similar layout (see below).
The specific controls for each channel on this page are described below.
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Zero Cross edit box and spin control
Here the zero cross level of the Constant Fraction Discriminator (CFD) for the Input Channels 1…4 can be set.
Units are millivolts (mV), the permitted range is 0 to 40. Type the value as an integer in the edit box and press
<Enter> or click on Apply. Alternatively, use the spin control (next to it) to increment / decrement the current
value. In this case changes take effect immediately without pressing <Enter>.
Discriminator Level edit box and spin control
Here the discriminator level of the Constant Fraction Discriminator (CFD) for the respective input channel can
be set. Units are millivolts (mV), the permitted range is 0 to 1,000. This corresponds to real input voltage levels
of 0 to −1,000 mV. Type the value as an integer in the edit box and press <Enter> or click on Apply.
Alternatively, use the spin control (next to it) to increment / decrement the current value. In this case changes
take effect immediately without pressing <Enter>.
Offset edit box and spin control
Shifts the relative timing of photon events and is designed to compensate optical and electrical delays (e.g. in
cables and optical paths), e.g. in order to shift your fluorescence decay within the sync frame so that it is not
truncated. This feature completely eliminates the need for adjustable delay boxes.
Units are picoseconds (ps), the permitted range is from −99,999 ps to +99,999 ps. Type the value as an integer
in the edit box and press <Enter> or click on Apply. The offset value can be entered in steps of picoseconds.
Alternatively, use the spin control next to the edit box to increment ⁄ decrement the current value. In this case
changes take effect immediately without pressing <Enter>.
All=Ch1 group of tickboxes
On the right hand side of the regular controls there is a special group "All=Ch1" with three tick boxes labelled
"ZeroCr.", "Disrc." and "Offset". When one of these is ticked, control of this parameter is coupled to the
corresponding input setting for channel 1. All other channels will automatically take the same setting. This
serves as a means of manipulating all channels at once, which can be helpful in the initial setup phase when
there are a lot of channels that must be brought to identical standard settings. Later, when individual fine tuning
is required, the tick can be removed and the individual channels can then be programmed individually again.
Note that removing the tick does not bring the previous individual settings back. If you have lost some specially
optimized settings you may load a data file with known good settings which will update the control panel
accordingly. It is therefore a good idea to save a data file after optimizations have been completed and name it
descriptively as something like "initial settings for experiment X".
6.4.3. Inputs 5…8
This page only exists when more than 4 input channels are installed. It has the same controls as the page for
inputs 1...4 and follows the same logic of operation (as described above).
6.4.4. Acquisition
Resolution edit box and spin control
Use this set of input controls to specify the acquisition resolution. Units are Picoseconds (ps). Possible choices
are the base resolution (1 ps) and binary multiples of this resolution (1, 2, 4, 8, 16, … ps). Type the desired
resolution value (ps) as an integer in the edit box and press <Enter> or click on Apply. Alternatively, use the
spin control (next to it) to increment / decrement the current value. In this case, changes take effect
immediately without pressing <Enter>. In case of entering values other than valid resolutions, the next
suitable resolution step is chosen automatically.
There are always 216 = 65,536 channels in one histogram. With the different resolutions, the approximate time
ranges covered are between 65,536 * 1 ps = 65.5 ns up to 65,536 * 33,554,432 ps = 2.199 s. The choice of
range must compromise between resolution and time span covered. The smallest range offers the best
resolution and the shortest span (vice versa for the largest range). For sync rates > 20 MHz the highest
resolution is usually most appropriate, since the smallest range still covers the full sync period. For lower sync
rates the histogram range is too small to cover the full sync period. The decay curve region of interest may
therefore lie outside the acquisition window of 65,536 time bins. Apart from switching to a lower resolution it is
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possible to shift the acquisition window relative to the sync frame by means of the offset. Note that working with
very long time spans (low sync rates) also requires long acquisition times to mimnimize noticeable “steps” in
the acquired histograms.
Acquisition Time edit box and spin control
Set the desired measurement time here. Units are Seconds (s). The permitted range is 0.001 to 360,000 in
steps of 0.001. Type the desired value in the edit box and press <Enter> or click on Apply. Alternatively, use
the spin control (next to it) to increment / decrement the current value. In this case, changes take effect
immediately without pressing <Enter>. The default increment / decrement per mouse click is logarithmic.
Offset edit box and spin control
For sync rates > 20 MHz the highest resolution is usually most appropriate, since the smallest range still covers
the full sync period. The offset should in these cases always be set to 0.
For lower sync rates the histogram range may be too small to cover the full sync period. The decay curve
region of interest may therefore lie outside the acquisition window of 65,536 channels. Apart from switching to
a lower resolution it is possible to shift the acquisition window relative to the sync frame by means of setting the
offset > 0.
Use this set of input controls to specify the desired offset. Units are Nanoseconds (ns). Possible offsets are in
the range from 0 to 500 ns. Type the value as an integer in the edit box and press <Enter> or click on Apply.
The offset value can be entered in steps of nanoseconds. Alternatively, use the spin control next to the edit box
to increment ⁄ decrement the current value. In this case changes take effect immediately without pressing
<Enter>.
Internally the offset is subtracted from each start–stop measurement before it is used to address the histogram
channel to be incremented. Therefore, increasing the offset means shifting the signal on the screen to the left,
towards earlier times.
Note that the offset has no effect on the relative timing of laser pulses and photon events. It merely shifts the
region of interest where data is to be collected. The relative timing of laser pulses and photon events can only
be controlled by means of cable delays (e.g. in order to shift your fluorescence decay within the sync frame so
that it is not truncated).
Trc./Block trace colour indicator, edit box and spin control
The HydraHarp can measure histograms in up to 512 memory blocks. Out of these, up to 8 curves can be
displayed and one "active block" can be used for measurements. Choose the active memory block you wish to
use for the next measurement here. The trace mapping dialog is used to select the curves for display. Make
sure the curve you are using is switched on (shown). You can open this dialog directly by clicking on the trace
colour indicator. The colour indicator shows the trace colour the chosen block is currently mapped to. If it is a
solid square, the curve is mapped and shown. If it is mapped but not shown, the indicator shows a small striped
square. If the curve is not even mapped for display the indicator remains white. Make sure not to overwrite
existing data in a block that was used before. In order to warn you if a trace is used, the heading of the
Trc/Block selector will turn red.
Restart check box
If this box is ticked (checked) the measurement will automatically restart after the acquisition time has elapsed.
Toggle the current setting with a mouse click. The setting is without effect in TRES mode.
Stop on Overflow check box
If this box is ticked (checked) the measurement will stop on overflow of any histogram bin. The overflow limit is
set by the "Stop At" parameter. Toggle the current setting with a mouse click.
Mode selection box
You may here select between three different acquisition modes:
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Oscilloscope Mode:
The selection Oscilloscope from the selection box "Mode" selects the current acquisition mode
Oscilloscope for the HydraHarp. In oscilloscope mode the acquisition and display, once started, repeats at
intervals given by the current acquisition time setting. The histogram accumulation always starts from
scratch. This is useful for monitoring fast changes during optical alignment etc.
Integration Mode:
As opposed to oscilloscope mode, the histogram acquisition in integration mode is not reset to zero with
each display refresh. The histogram continues to grow while the display is updated every 0.1 to 1 seconds
determined by the refresh rate value selected in the General Settings Dialog. Acquisition can be manually
started and stopped, additionally the option "Stop on Overflow" will stop the acquisition when the maximum
count of any channel overflows. The option "Restart" will cause the acquisition to start again after the
acquisition time has elapsed. This is similar to oscilloscope mode but allows watching the histogram grow if
the acquisition time is fairly long.
TRES Mode:
Selects the measurement mode TRES (Time–Resolved Emission Spectra). This requires a monochromator and corresponding driver hardware / software to be installed. See section 5.3.7.
Stop At edit box and spin control
Here the stop count level for histogramming can be set. The setting is only meaningful if the check box
"Stop Ofl" is active. Type the value as an integer in the edit box and press <Enter> or click on Apply.
Alternatively, use the spin control (next to it) to increment / decrement the current value. In this case changes
take effect immediately without pressing <Enter>. The minimum is 0. If the entered value exceeds the allowed
maximum of 4,294,967,295, an error message appears. When using the spin control exceeding the maximum
is simply not allowed. Instead of incrementing the value further, the system beeps a warning.
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6.5. Axis Panel
The controls of the Axis Panel are used to customise the main window's curve display.
Apply–button
All mouse actions on the spin controls on this dialog result in instant modification of the display. If you choose
to alter the values in the edit fields by keyboard, you have to finish your changes by pressing <Enter> or to
click the Apply–button.
All other controls are grouped as follows:
6.5.1. Time Axis Group
Note that the time axis labelling is precise in terms of relative information only. At offset=0 the absolute time
difference between the inputs is close to the shown times but may be off by some tens of picoseconds. Offsets
>0 change the placement of the acquisition window and the true time differences are then off accordingly.
Minimum edit box and spin control
Here the start of the displayed time axis can be set. Units are nanoseconds (ns). The minimum is 0. If the
entered value exceeds the current time axis maximum, an error message appears. When using the spin control
exceeding the current time axis maximum is simply not allowed. Instead of incrementing the minimum value
further, the system beeps a warning. Observe the notes on time axis interpretation given in the paragraph Time
Axis Group above.
Maximum edit box and spin control
Here the end of the displayed time axis can be set. Units are nanoseconds (ns). The maximum allowed here is
2.2 s. If the entered value becomes smaller than the current time axis minimum, an error message appears.
When using the spin control, violating the current time axis minimum is automatically prevented. Instead of
decrementing the minimum value further, the system beeps a warning. Observe the notes on time axis
interpretation given in the paragraph Time Axis Group above.
The time axis settings will determine the number of time bins exported via clipboard copy / paste. Only the bins
within the displayed time range will be used.
6.5.2. Count Axis Group
Minimum edit box and spin control
Here the start of the displayed count axis can be set. Units are counts. In linear scale mode the default
increment / decrement is 100. In log scale mode it is one decade. If the minimum is set to 0 in logarithmic
mode, the resulting minimum as displayed is 10−1. If the entered value exceeds the current count axis
maximum, an error message appears. When using the spin control, exceeding the current count axis maximum
is automatically prevented. Instead of incrementing the minimum value further, the system beeps a warning.
Maximum edit box and spin control
Here the upper end of the displayed count axis can be set. Units are counts. In linear scale mode the default
increment / decrement is 100. In log scale mode it is one decade. If the entered value becomes smaller than
the current time axis minimum, an error message appears. When using the spin control, violating the current
count axis minimum is prevented automatically. Instead of decrementing the minimum value further, the system
beeps a warning.
Lin / Log radio buttons
These radio buttons change the inner scaling of the count axis from linear to logarithmic and vice versa.
Despite the fact that in logarithmic scale the display will always show whole decades, the change of the scaling
is applied without modifying the minimum / maximum range.
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6.6. Trace Mapping Dialog
The HydraHarp software can measure histograms in up to 512 memory blocks. Out of these, up to 8 curves
can be displayed. The trace mapping dialog is used to select the curves to display. Tick the individual boxes
‘Show’ to display a curve. Select the number of the memory block you wish to map the individual display curve
to. The Trace Mapping Dialog also provides some statistics on each curve. These items are:
FWHM The Full Width Half Maximum of the curve peak (usually for IRF traces)
Max Count The count in the highest point of the curve
At Time The time corresponding to the Max Count channel
Resolution The channel width (time bin width) of the curve
Furthermore there are several buttons:
Details Can be clicked to see more curve information
All Can be clicked to tick all traces as shown
0..7 Can be clicked to set the default mapping of trace 0..7 to block 0..7
Clear (trash can) Can be clicked to delete the contents of individual blocks
The trace mapping dialog can be launched from the corresponding button on the toolbar as well as through the
trace colour indicator on the control panel.
6.7. General Settings Dialog
Use this dialog to change standard settings of the HydraHarp software. Notably these are: Prompt overwrite
(warning before overwriting existing data) and TTTR Marker Settings. The control connector of the HydraHarp
400 provides TTL inputs for synchronization signals. The markers can be enabled resp. disabled and recorded
at the rising or falling edge of the corresponding TTL signal. The active edges can be chosen here. All settings
will be kept in the Windows registry and will be retrieved at the next program start. T hey will be individual to
each user.
The dialog's controls are grouped as follows:
Display Group
Display Rate /s Sets the time interval between display updates from (0.1s to 1s).
Draw mode Switches between different curve draw–modi (Lines / Stairs).
Grid Checked: The curve display is grated with a light–grey grid
Unchecked: The axes of the curve display are marked with ticks
File Saving Group
Prompt overwrite Activate this check box if you wish to be prompted before saving data over
existing data in a file (recommended).
Warnings Group
Here you can selectively enable/disable individual warnings. Activate the check box for each warning you wish
to receive. See section 8.1 for details.
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TTTR Marker Settings Group
Here you can set the signal specifications for external marker signals used in TTTR mode (T2 and T3 mode).
The settings for the different markers are independent from each other. The following table applies to each of
the four markers:
enable Check this if you want the marker to be included into the TTTR data
stream.
rising / falling Radio buttons to identify the active edge of the signal.
Reference Clock Source Group
The setting in this group takes effect after the next software start.
internal / external Select whether the HydraHarp uses its internal clock or an external 10 MHz
clock supplied at the REF IN connector.
6.8. About HydraHarp… Dialog
This dialog provides version information on the HydraHarp software and hardware, the latter only if it is
installed an operational. It can be opened via the Help menu or via the toolbar. The button Request Support
opens a small text viewer window that provides a listing of HydraHarp hardware and software versions that you
can copy and paste into your support enquiry. Note that this information is very important for adequate support.
Support requests without this information cannot be processed and will be delayed by return questions for this
information. If your system is not functional at all, the minimum information you must provide for support is the
serial number of your HydraHarp. It can be found at the back of the housing. The dialog also provides buttons
for links to relevant web pages and a button for download of software updates. Note that downloading a
software update does not automatically install it. Typically the downloads contain zip files that must be
unpacked and installed manually. Also note that it may not always be advisable to blindly install the latest
software. This is the case especially when a user or his/her laboratry have developed custom software that
relies on a certain file format. Updates may break such compatibility. Therefore, always study the release notes
before installing.
6.9. Title and Comment Editor
You can use this dialog to edit the file title / comment. It can be opened via double–click on the title, via the File
menu or via the Print Preview Toolbar. The text you enter here will be stored in the data file. The first line will
be displayed as the file title over the histogram display area. The text you can enter here is limited to 4 lines
and 255 characters. Upon loading of a data file, the title and comment will also be retrieved. It will also be
included in prints, if the corresponding check mark is set in the page setup dialog box available through the
File menu.
6.10. Print Preview Dialog
Use this Dialog to preview the layout as it would appear when printed. When you choose this command, the
main window will be replaced with a print preview window in which one or two pages will be displayed in their
printed format. The print preview dialog offers the following options:
Print
Bring up the print dialog box, to start a print job.
Next Page
Preview the next printed page.
Prev Page
Preview the previous printed page.
One Page / Two Page
Preview one or two printed pages at a time.
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Zoom In
Take a closer look at the printed page.
Zoom Out
Take a larger look at the printed page.
Page Setup
Change the layout of the printed page.
Close
Return from print preview to the editing window.
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7. Problems, Tips & Tricks
7.1. PC Performance Issues
The HydraHarp device and its software interface are a complex real–time measurement system demanding
appropriate performance both from the host PC and the operating system. This is why a fairly modern CPU and
sufficient memory are required. The screen resolution should be at least 800x600. At least a 1 GHz processor,
1 GB of memory and a fast hard disk are recommended.
In order to maintain correct interaction between the measurement hardware and the display of histogram
curves and user input, the operating system’s message passing mechanism is used. It is recommended not to
overload the system by running other processes in the background while measuring with the HydraHarp. The
PC’s own occasional network activity should be no problem but running the machine e.g. as a server for other
PCs is not recommended. In principle, of course, any kind of background activity is allowed. Should the system
become overloaded the HydraHarp histograms will not be "wrong" or distorted, but measurement times and
display rates may become irregular. You can even iconize the HydraHarp during measurements without doing
damage. This may be of interest for lengthy measurements in integration mode, where one is only interested in
accumulating a certain amount of counts without need for permanent monitoring. The "panel meters" showing
the current count rates are not meant to be 100 % accurate. They merely serve as an aid for setting up the
system. Some of them may suffer from system overload. Accordingly the values shown in the curve details
(trace mapping dialog) are subject to such tolerances.
In TTTR mode, system overload will manifest itself in loss of data. Here no other background processing
should be allowed. The faster the PC, the less these issues will matter. TTTR mode performance (i.e. max.
throughput without loss) may also be improved by de–fragmenting the hard disk before long measurements.
7.2. USB interface
In order to deliver maximum throughput, the HydraHarp 400 uses (dependent on the model you purchased)
USB 2.0 or USB 3.0 bulk transfers. Correspondingly, the HydraHarp must rely on having a suitable host
interface of appropriate speed rating. USB 2.0 host controllers of modern PCs are usually integrated on the
mainboard. Unless your PC is fairly new, USB 3.0 interfaces may need to be upgraded as PCIe cards.
Throughput is usually limited by the host controller and not by the HydraHarp. Make sure that the host
controller drivers are correctly installed and up to date. This may require using the mainboard manufacturer's
driver disk rather than relying on Windows drivers. Note that some of the early USB 3.0 host controllers still
have immature drivers and possibly flawed firmware. Check the mainboard manufacturer's website for updates
if you encounter problems. For best performance, do not run other bandwidth demanding devices on the same
USB interface when working with the HydraHarp. USB cables must be rated for the intended USB speed. Older
cables do not meed this requirement and can lead to errors and malfunction. Similarly, some PCs have poor
internal USB cabling, so that USB sockets at the front of the PC are often unreliable.
7.3. Histogram Artefacts
Disturbing histogram ripple is strongly dependent on the quality of the input signals. Try to deliver the best
possible signal quality with clean and steep active edges and without overshoot and ringing. It is recommended
to use detector and sync signals of similar amplitude to avoid excess crosstalk. If the PDL 800–B picosecond
diode laser is used, attenuating the sync pulses by 10 dB immediately at the laser driver may reduce histogram
ripple (set CFD level accordingly). Always use good quality 50 Ω components and coax leads with proper
shielding and correct termination. We recommend cables of type RG223 with double braided and silver plated
screen. Check your setup for ground loops. Grounding different system components (PC, detector, detector
power supply, diode laser driver, etc.) at different points can induce considerable noise in the ground lines.
Because signal return paths may share the same ground lines, this noise is copied into the signal lines and
causes increased timing jitter and / or histogram ripple. Network cables and mobile phones may also be
sources of surprising noise. Use properly impedance matched power splitters (reflection–free T–pads) if
signals must be fed to multiple 50 Ω inputs. Never use ordinary Tees. All accessories are available from
PicoQuant. PMT detectors should be connected through a suitable high speed preamplifier (available from
PicoQuant). MCP–PMT detectors should be connected through an amplifier with slightly higher gain (e.g.
PAM 102–M from PicoQuant). TTL–SPAD–detectors (e.g. Excelitas SPCM–AQR) must be connected through a
pulse inverter (PicoQuant SIA 400).
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A problem often overlooked is pile–up and dead time related histogram distortion. It becomes noticeable if the
detector count rate exceeds 2 % of the sync rate. This is why high excitation rates are so important. Pile–up is
an inherent problem of TCSPC and not a fault of the hardware. The only way to handle it safely is to maintain
count rates < 1% to 2 % of the sync rate. Pile–up may to some extent also be corrected for, but this can be
extremely difficult in practice. For further details see our TechNote on TCSPC or the literature given in the end
of section 2.4.
7.4. Warming Up and Calibration
Observe the warm–up period of at least 20 minutes (dep. on ambient temperature) before using the HydraHarp
for serious measurements. Warming up takes about 20 to 30 minutes dependent on the lab temperature. It is
completed when the cooling fan starts to run for the first time. You can use this time for set –up and preliminary
measurements. Calibrate one more time before starting serious measurements. The calibration takes only a
few seconds. For very long measurements allow some more time for thermal stabilization and avoid
temperature changes > 5 K in the environment of the instrument. The permissible ambient temperature is 15°C
to 35°C. Do not obstruct the cooling fan at the back and the air inlets at the bottom of the housing. Make sure
that the cooling air can circulate freely and no other hot instrument is under the cooling inlets.
7.5. Custom Programming of the HydraHarp
A programmer's library (DLL) for custom Windows software development is available to build your own
applications e.g. in LabVIEW, Matlab, C++, C#, Delphi, Lazarus or Visual Basic. A rich set of demo code is
provided for an easy start. A Library for Linux is also available. It provides the same interface so that
applications can easily be ported between the platforms.
7.6. Software Updates
We constantly improve and update the software for our instruments. This includes updates of the configurable
hardware (FPGA). Such updates are important as they may affect reliability and interoperability with other
products. The software updates are free of charge, unless major new functionality is added. It is strongly
recommended that you register for email notification on software updates. If you wish to receive such
notification, please email your name and the serial number of your PicoQuant product(s) to
info@picoquant.com. You will then receive update information with links for download of any new software
release. Alternatively you can click the button 'Check for Updates' in the Help-About dialog available through
the main menu or the toolbar. Note that downloading a software update by means of this button does not
automatically install it. Typically the downloads contain zip files that must be unpacked and installed manually.
Also note that it may not always be advisable to blindly install the latest software. This is the case especially
when a user or his/her laboratry have developed custom software that relies on a certain file format. Updates
may break this compatibility. Therefore, always study the release notes before installing updates.
7.7. Support and Bug Reports
The HydraHarp 400 TCSPC system has gone through extensive testing. Nevertheless, it is a very cutting edge
product and we would like to offer you our support in any case of problems with the system. Do not hesitate to
contact PicoQuant in case of difficulties with your HydraHarp.
As a first step it is always advisable to study the manual or pres the help button. As a second step you may
want to consult PicoQuant's user forum at http://forum.picoquant.com/ where a similar issue may already be
described.
Should you observe errors or bugs caused by the HydraHarp system please try to find a reproducible error
situation. Then open the About HydraHarp dialog. This dialog provides version information on the HydraHarp
software and hardware (the latter only if it is installed and operational). It can be opened via the Help menu or
via the toolbar. The button Request Support opens a small text viewer window that provides a listing of
HydraHarp hardware and software versions that you can copy and paste into your support enquiry. Note that
this information is very important for adequate troubleshooting. Support requests without this information
cannot be processed and will be delayed by return questions for this information. If your system is not
functional at all, the minimum information you must provide for support is the serial number of your HydraHarp.
It can be found at the back of the housing. There may be other relevant circumstances, especially other new
hardware installed in your PC, so please provide details. You can run msinfo32 to obtain a listing of your PC
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configuration and attach the summary file to your error report. E–mail this complete set of information to
support @picoquant.com. Your feedback will help us to improve the product and documentation.
If you have serious problems that require the device to be sent in for inspection / repair, please request an
RMA number before shipping the hardware. Observe precautions against static discharge and physical
damage under all circumstances in handling, packaging and shipping. Use original or equally protective
packaging material.
Of course we also appreciate good news: If you have obtained exciting results with one of our systems, please
let us know, and where appropriate, please mention the instrument in your publications. The simplest way of
doing this in case of the HydraHarp 400 is citing the original publication about the instrument1. At our web–site
we also maintain a large bibliography of publications referring to our instruments. It may serve as a reference
for you and other potential users. See http://www.picoquant.com/scientific/references. Please submit your
publications for addition to this list.
1 Wahl M., Rahn H.-J., Röhlicke T., Kell G., Nettels D., Hillger F., Schuler B., Erdmann R.: Scalable timecorrelated photon counting system with multiple independent input channels. Review of Scientific Instruments,
Vol.79, 123113 (2008)
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8. Appendix
8.1. Warnings
When the HydraHarp software is running with functional hardware it continuously collects
information about the input signals and the current acquisition settings. If these settings together
with the input rates indicate possible errors, the software will indicate this by means of a warning
icon.
While the software is running in interactive histogramming mode the warning icon is displayed at the bottom of
the main window in the status bar. In the case of TTTR mode, it will appear directly in the TTTR mode dialog.
The icon can be clicked to display a list of current warnings together with a brief explanation of each warning.
Note that the software can detect only a subset of all possible error conditions. It is therefore not safe to
assume “all is right” just by seeing no warning. On the opposite, if any of the warnings turns out to be an
unnecessary nuisance, e.g. because your specific measurement conditions knowingly force it, you can disable
that warning via the general settings dialog (see section 6.7).
The warnings are to some extent dependent on the current measurement mode. Not all warnings will occur in
all measurement modes. Also, count rate limits for a specific warning may be different in different modes. The
following table lists the possible warnings in the three measurement modes and gives some explanation as to
their possible cause and consequences.
Warning Histo Mode T2 Mode T3 Mode
WARNING_SYNC_RATE_ZERO
No counts are detected at the sync input. In histogramming and
T3 mode this is crucial and the measurement will not work
without this signal. Note that at very low sync rates this warning
may occur spuriously due to the rate meter limitations. You may
want to switch it off in this case.
√ √
WARNING_SYNC_RATE_TOO_LOW
This warning should not occur with current software and
firmware. It is kept in this list for backward compatibility only.
Note, however, that there actually is a minimum sync rate of
about 0.25 Hz in histogramming and T3 mode. Due to the lower
limit of the rate meters this condition cannot be detected, hence
no warning is issued.
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Warning Histo Mode T2 Mode T3 Mode
WARNING_SYNC_RATE_TOO_HIGH
In histogramming or T3 mode:
The divided pulse rate at the sync input is higher than 12.5 MHz
(deadtime limit). Use a larger divider if possible.
In T2 mode:
The pulse rate at the sync input is higher than 9 MHz (bus limit if
running at USB 2.0 speed) or higher than 12.5 MHz (deadtime
limit if running at USB 3.0 speed). The measurement will
inevitably lead to a FiFo overrun. The most common reason for
this error is that a laser sync signal is still connected. T2 mode is
normally intended to be used without a sync signal. There are
some rare measurement scenarios where this condition is
expected and the warning can be disabled. Examples are
measurements where the FiFo can absorb all data of interest
before it overflows.
√ √ √
WARNING_INPT_RATE_ZERO
No counts are detected at any of the input channels. In
histogramming and T3 mode these are the photon event
channels and the measurement will yield nothing. You might
sporadically see this warning if your detector has a very low dark
count rate and is blocked by a shutter. In that case you may want
to disable this warning.
√ √
WARNING_INPT_RATE_TOO_HIGH
You have selected T2 or T3 mode and the overall pulse rate at
the input channels is higher than the USB speed limit (9 MHz with
USB 2.0 connection or 40 MHz with USB 3.0 connection). The
measurement will inevitably lead to a FiFo overrun. There are
some rare measurement scenarios where this condition is
expected and the warning can be disabled. Examples are
measurements where the FiFo can absorb all data of interest
before it overflows.
√ √
WARNING_INPT_RATE_RATIO
This warning is issued in histogramming and T3 mode when the
rate at any input channel is higher than 5% of the sync rate. This
is the classical pile-up criterion. It will lead to noticeable deadtime artefacts. There are rare measurement scenarios where this
condition is expected and the warning can be disabled. Examples
are antibunching measurements.
√ √
WARNING_DIVIDER_GREATER_ONE
You have selected T2 mode and the sync divider is set larger
than 1. This is probably not intended. The sync divider is
designed primarily for high sync rates from lasers and requires a
fixed pulse rate at the sync input. In that case you should use T3
mode. If the signal at the sync input is from a photon detector
(coincidence correlation etc.) a divider > 1 will lead to unexpected
results. There might be some rare measurement scenarios where
this condition is intentional and the warning can be disabled.
√
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Warning Histo Mode T2 Mode T3 Mode
WARNING_TIME_SPAN_TOO_SMALL
This warning is issued in histogramming and T3 mode when the
sync period (1/SyncRate) is longer that the start to stop time span
that can be covered by the histogram or by the T3 mode records.
You can calculate this time span as follows:
Histogramming mode: Span = Resolution * 65536
T3 mode: Span = Resolution * 32768
Events outside this span will not be recorded. There are some
measurement scenarios where this condition is intentional and
the warning can be disabled.
√ √
WARNING_OFFSET_UNNECESSARY
This warning is issued in histogramming and T3 mode when an
offset >0 is set even though the sync period (1/SyncRate) can be
covered by the measurement time span without using an offset.
The offset may lead to events getting discarded. There are some
measurement scenarios where this condition is intentional and
the warning can be disabled.
√ √
If any of the warnings you receive indicate wrong pulse rates, the cause may be inappropriate input settings,
wrong pulse polarities, poor pulse shapes or bad connections. If in doubt, check all signals with an oscilloscope
of sufficient bandwidth.
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8.2. Data File Formats
While for many purposes the ASCII export of histograms to files or to the clipboard is sufficient and easy, you
also may want to access the HydraHarp data files via custom programs. This section provides only a brief
overview on the file format. For details please refer to the online help file available via the help menu.
To overcome certain limitations of the formats used in the past, the latest HydraHarp software uses a new file
format previously proven in our SymPhoTime 64 software. It is designed to be future poof in the sense that files
created by the current software version stay valid for future software revisions and, moreover, files created by
future software versions will most likely still be readable by older software, although they might contain
information, that software can't even "know" about. This is achieved by using a tagged format. Tags identify the
data to follow, and give the type, length and even meta information. The exact location of an individual item in
the file is then irrelevant. Version robustness is granted as long as version-breaking changes to the semantics
of a given field are implemented by a tag with a new identifier rather than expanding the range or interpretation
of the old one. The list af tags (identifiers) and their interpretation rules can be kept in a tag dictionary. With this
as a precondition, the software only has to show tolerance on missing non-mandatory (i.e. optional) content.
The new format definition unifies PicoQuant's existing file formats which individually evolved over many years.
The resulting new TTTR file format with the extension *.ptu will be used for all current and future TCSPC
products supporting TTTR mode and enriches them by powerful new features. Similarly, a tagged file format
with the extension *.phu will cover the histogram data formats of our current and future TCSPC products.
The new format may at first glance look more complicated than previous versions with hard coded structure.
Nevertheless, we hope that the benefits of version tolerance and “durability” will outweigh the complications. To
support understanding of the format and implementation of custom software acessing these files, a set of
demos is provided in the subfolder \Filedemo in your chosen installation folder. If you need to evaluate
more header items than the demos do, please refer to the HydraHarp online help file available via the help
menu. A file format related html help file is also provided in the file demo folder. It contains a list of tag types
and a tag dictionary that explains the individual items. Note that the dictionary contains more items than the
HydraHarp software actually uses. It is recommended to go by a specific file, have one of the demos read it
and then look at the list of header items you get. You can also use the PicoQuant File Info shell extension that
will be installed by the HydraHarp software setup to inspect individual header items of a *.ptu or *.phu file.
Just right-click on the file in Windows explorer and select Properties.
For backward compatibility version 3.0 of the HydraHarp software can still read old files of format version 2.0
(*.hhd, *.ht3). Note, however, that saving such files converts them to the new format. Despite the intended
version tolerance of the tagged format, for consistency and safe version checking the new HydraHarp data
files still carry a format version number, which is now called content version and currently has the string value
“3.0”. In order to identify a HydraHarp data file as a file created by and to be used by the native HydraHarp
software there is a tag assured content which begins with the string “HydraHarp“. There is also a pair of tags
creator name and creator version that identify the creating software. Programmers of custom software writing
such files MUST USE THEIR OWN CREATOR NAME.
Note that despite our best efforts towards version tolerance, file formats in future software releases are subject
to change without notice.
8.2.1. Interactive Histogramming Mode File Format
The standard HydraHarp histogram data files created by the HydraHarp software in interactive histogramming
mode (*.phu) are tagged binary files which contain both the setup parameters and the actual histogram data.
The latter can be present multiple times, i.e. multiple measurements can be stored in one file. Relevant settings
are stored for each meaurement separately. In order to identify a HydraHarp data file as a file created by and to
be used by the native HydraHarp software, a program reading in these files can read the tag assured content
which begins with the string “HydraHarp“. However, a piece of software aiming solely at retrieving the
histogram data content can (and should) be tolerant about this tag and go for the pure histogram data. This
tolerance will ensure compatibility for the future. Indeed, the demos in the subfolder \Filedemo in your
chosen installation directory are following this tolerant approach. For more information on individual file tags
and their content, please consult the online help file available via the help menu.
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8.2.2. TTTR Mode File Format
HydraHarp data files from T2 and T3 Mode (*.ptu) created by the HydraHarp software are tagged binary files
which contain both the setup parameters and the actual event data. There can be only one measurement per
file. The setup data in the file header which is similar to that in standard interactive mode files. In order to
identify a HydraHarp data file as a file created by and to be used by the native HydraHarp software, a program
reading in these files can read the tag assured content which begins with the string “HydraHarp“. However, a
piece of software aiming solely at retrieving the histogram data content can (and should be) be tolerant about
this tag and go for the pure event record data. This tolerance will ensure compatibility for the future. Indeed, the
demos in the subfolder \Filedemo in your chosen installation directory are following this tolerant approach.
For more information on individual file tags and their content, please consult the online help file available via
the help menu. It is worth noting that the actual TTTR record data following the file header has the same
structure as that of the previous file format version 2.0 (*.ht2,*.ht3) and corresponds directly to the raw
data obtained with custom programs using the HydraHarp programming library.
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8.3. Hardware Technical Data
8.3.1. Specifications
All information given here is reliable to our best knowledge. However, no responsibility is assumed for possible
inaccuracies or omissions. Specifications and external appearance are subject to change without notice.
Input Channels and Sync
Trigger principle Vertex Finding CFD
software adjustable
Trigger point Falling edge (negative going)
Input voltage operating range (pulse peak, 50 Ω)
optimum:
0 mV … −1 000 mV
−100 mV … −500 mV
Input voltage max. range (damage level) ± 1 500 mV
CFD level adjust 0 mV … −1 000 mV
CFD zero cross adjust 0 mV … 40 mV
Trigger pulse width 0.5 ns … 30 ns
Trigger pulse rise ⁄ fall time ≤ 2 ns
Max. count rate
12.5 · 106 cps
Max. sync rate without divider 12.5 MHz
Max. sync rate with divider 1 ⁄ 16 150 MHz
Adjustable delay range for each input channel
resolution:
± 100 ns
1 ps
Marker and Control inputs standard TTL, 100 kΩ DC load
Time to Digital Converter
Base resolution (min. time bin width) 1 ps
Timing uncertainty (rms)
< 12 ps + 10−8 · Δt
Differential non–linearity (full measurement range) < 2.0% p.p.
< 0.2% rms
Dead time < 80 ns
External Reference Clock
Input 10 MHz
200 mV … 1 000 mV p.p.
50 Ω; AC coupled
Output 10 MHz
300 mV p.p.
50 Ω; AC coupled
Histogrammer
Maximum number of time bins 65 536
Bin widths 1, 2, 4, 8, …, 33 554 432 ps
Measurement ranges 65 536 x 1 ps … 65 536 x 33.55 µs
Count depth per time bin 4 294 967 296 (32 bit)
Acquisition time 1 ms … 100 h
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TTTR Engine
T2 mode resolution 1 ps
T3 mode resolution 1, 2, 4, 8, …, 33 554 432 ps
FIFO buffer depth (records) 2 097 152
Sustained throughput limit (sum of all channels)*
* depends on host PC configuration and performance
USB 2.0 typ. 9 · 106 events ⁄ s
USB 3.0 typ. 40 · 106 events ⁄ s
Acquisition time 1 ms … 100 h
External Marker input pulse duration > 50 ns
External Marker time recording error in T2 mode < 100 ns
External Marker time recording error in T3 mode < Tsync + 100ns
Operating Environments
Ambient temperature 15°C … 35°C
59°F … 95°F
Warm–up period ≥ 20 min
Bus interface Model dependent: USB 2.0 or USB 3.0
Recommended PC specs
CPU clock
RAM
≥ 1 GHz
≥ 1 GB
Operating system Windows 7 ⁄ 8 ⁄ 8.1
Power Consumption
Line 100 V … 240 V AC small frame ≤ 50W, large frame ≤ 100W
Dimensions
Small frame (up to 4 channels) 310 × 235 × 140 mm
Large frame (up to 8 channels) 310 × 340 × 140 mm
Retraction of old Devices
Waste electrical products must not be
disposed of with household waste.
This equipment should be taken to your
local recycling centre for safe treatment.
WEEE–Reg.–Nr. DE 96457402
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8.3.2. Connectors
Apart from the SMA connectors for the input signals the HydraHarp 400 provides several connectors for other
purposes such as experiment control. The following table lists their function and signal types, sorted by the
module they belong to. For details on the signal specifications (voltages, pulse widths etc.) see section 8.3.1.
All SMA connectors are designed for 50 Ohms coaxial cable connections and are terminated accordingly. All
Lemo connectors are for high impedance TTL signals and not terminated. In case of TTL outputs they must
never be connected to loads with 50 Ohms termination.
HDU 404 (CONTROL)
Connector Label Connector Type Signal Type Function
USB 2.0 USB 2.0 Type B
receptacle
USB 2.0 host communication
CAN Sub–D 9 pin
male
CAN Bus control of external devices (proprietary)
HDU 504 (CONTROL)
Connector Label Connector Type Signal Type Function
USB 3.0 USB 3.0 Type B
receptacle
USB 3.0 host communication
HSR 110 (SYNC)
Connector Label Connector Type Signal Type Function
REF IN SMA female 10 MHz clockinexternal reference clock, e.g. from a frequency normal
or atomic clock, or from another HydraHarp 400
REF OUT SMA female 10 MHz clock
out
reference clock output e.g. for another HydraHarp 400
SYNC IN SMA female NIM in timing signal from sync source (typically a laser)
M1 ... M4 NIM-CAMAC
receptacle Lemo
ERN.00.250**
TTL in external markers for external device synchronization,
e.g. line and frame in image scanning
(select active edge and enabling via software)
C1 ... C2 NIM-CAMAC
receptacle Lemo
ERN.00.250**
TTL in experiment control (requires dedicated software)
A NIM-CAMAC
receptacle Lemo
ERN.00.250**
TTL out
(active low)
'active' signal for use by external hardware
(must not be loaded in excess of 10 mA)
TDC 210 (TIMING)
Connector Label Connector Type Signal Type Function
IN SMA female NIM in timing signal input (typically from a photon detector)*
ENA NIM-CAMAC
receptacle Lemo
ERN.00.250**
TTL in count enabling of the corresponding input*
(high or open = enabled, low = disabled)
*Note: The HydraHarp software enumerates the physical input channels as logical input channels 1 to N, where N depends on the number
of installed TDC modules. The mapping of physical to logical channels is obtained by counting as follows: start form the leftmost TDC
module and count top to bottom within each TDC module. At the time of factory assembly this mapping will be marked by plastic labels at
each IN connector.
** Suitable plugs from the Lemo catalog are FFA.00.250.xxx. Alternatively the BNC adaptor ABF.00.250.CTA can be used.
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8.3.3. Indicators
The HydraHarp 400 front panel provides several LED indicators signalling the instrument status. The following
table lists their function, sorted by the module they belong to. Note that precise status information should be
obtained from the software.
HDU 404 (CONTROL)
LED Label LED Function Colour Interpretation
POWER power status of entire instrument green = OK
dark = failure
STATUS status of control unit green = OK
red = permanent: error
(software will report details)
brief flash: transient state, no error
HDU 504 (CONTROL)
LED Label LED Function Colour Interpretation
POWER power status of entire instrument green = OK
dark = failure
STATUS status of control unit green = OK
red = permanent: error
(software will report details)
brief flash: transient state, no error
USB status of USB connection green = USB 3.0 super speed
orange = USB 2.0 high speed
red = no connection or error
flicker = data transfer in progress (note that fast
flicker may appear like orange light)
HSR 110 (SYNC)
LED Label LED Function Colour Interpretation
RDY status of sync unit green = OK
orange = thermal stabilization and subsequent
calibration not yet completed
red = error
(software will report details)
REF status of reference clock green = OK
red = error
(probably external reference missing)
TDC 210 (TIMING)
LED Label LED Function Colour Interpretation
RDY status of timing unit green = OK
orange = thermal stabilization and subsequent
calibration not yet completed
red = error
(software will report details)
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8.4. Using the Software under Linux
The HydraHarp software can also be used under Linux (x86 platform only). This requires that Wine is installed
(see http://www.winehq.org). We have successfully tested with Wine 1.7.11. You can run the regular software
setup as explained in section 3.2. Instead of installing a device driver, running under Linux with Wine requires
that you have Libusb 0.1 or better the combination of Libusb 1.0 and Libusb-Compat installed (see
http://libusb.sourceforge.net/).
Libusb Access Permissions
For device access through libusb, your kernel needs support for the USB filesystem (usbfs) and that filesystem
must be mounted. This is done automatically, if /etc/fstab contains a line like this:
usbfs /proc/bus/usb usbfs defaults 0 0
This should routinely be the case if you installed any of the mainstream Linux distributions.
The permissions for the device files used by libusb must be adjusted for user access. Otherwise only root can
use the device(s). The device files are located in /proc/bus/usb/. Any manual change would not be
permanent, however. The permissions will be reset after reboot or replugging the device. The proper way of
setting the suitable permissions is by means of hotplugging scripts or udev. Which mechanism you can use
depends on the Linux distribution you have. Most recent distributions use udev.
Hotplug
For automated setting of the device file permissions with hotplug you have to add an entry to the hotplug
“usermaps”. These files are located in /etc/hotplug/. If the entire hotplug directory does not exist, you
probably don't have the hotplug package installed. This may be because your distribution is rather old (you may
still be able to install hotplug) or it is fairly recent and uses udev instead of hotplug (see section below).
Every line in the hotplug usermap files is for one device, the line starts with a name that will also be used for a
handler script. Since there are two HydraHarp 400 models (USB 2.0 and USB 3.0) the HydraHarp usermap file
has two lines as shown below. Line wraps are due to limited page width.
HydraHarp400 0x0003 0x0e0d 0x0004 0x0000 0x0000
0x00 0x00 0x00 0x00 0x00 0x00 0x00000000
HydraHarp400 0x0003 0x0e0d 0x0009 0x0000 0x0000
0x00 0x00 0x00 0x00 0x00 0x00 0x00000000
The handler script “HydraHarp400” gets started when the device is connected or disconnected. It looks like
this:
#!/bin/bash
if [ "${ACTION}" = "add" ] && [ -f "${DEVICE}" ]
then
chown root "${DEVICE}"
chgrp users "${DEVICE}"
chmod 666 "${DEVICE}"
fi
When the HydraHarp gets switched on or connected, the hotplug system will recognize it and will run the script,
which will then change the permissions on the device file associated with the device to 666, which means that
everybody can both read and write from/to this device. Note that this may be regarded as a security gap. If you
wish tighter access control you can create a dedicated group for HydraHarp users and give access only to that
group.
Both a usermap file HydraHarp400.usermap and the handler script HydraHarp400 are provided in the
folder Linux/hotplug on the distribution media. Copying both of them to /etc/hotplug/usb/ should be
sufficient to set up acces control via hotplugging for your HydraHarp.
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Udev
For automated setting of the device file permissions with udev you have to add an entry to the set of rules files
that are contained in /etc/udev/rules.d. Udev processes these files in alphabetical order. The default file
is usually called 50-udev.rules. Don't change this file as it could be overwritten when you upgrade udev.
Instead, put your custom rule for the HydraHarp in a separate file. The contents of this file for the handling of
the current HydraHarp 400 models should be:
ATTR{idVendor}=="0d0e", ATTR{idProduct}=="0004", MODE="666"
ATTR{idVendor}=="0d0e", ATTR{idProduct}=="0009", MODE="666"
A suitable rules file HydraHarp.rules is provided in the folder Linux/udev on the distribution media. You can
simply copy it to the /etc/udev/rules.d folder. The install script in the same distribution media folder does
just this. Note that the name of the rules file is important. Each time a device is detected by the udev system,
the files are read in alphabetical order, line by line, until a match is found. Note that different distributions may
use different rule file names for various categories. For instance, Ubuntu organizes the rules into further files:
20-names.rules, 40-permissions.rules, and 60-symlinks.rules. In Fedora they are not separated
by those categories, as you can see by studying 50-udev.rules. Instead of editing the existing files, it is
therefore usually recommended to put all of your modifications in a separate file like 10-udev.rules or 10-
local.rules. The low number at the beginning of the file name ensures it will be processed before the
default file. However, later rules that are more general (applying to a whole class of devices) may later override
the desired acecss rights. This is the case for USB devices handled through Libusb. It is therefore important
that you use a rules file for the HydraHarp that gets evaluated after the general case. The default naming
HydraHarp400.rules most likely ensures this but if you see problems you may want to check.
Note that there are different udev implementations with different command sets. On some distributions you
must reboot to activate changes, on others you can reload rule changes and restart udev with these
commands:
# udevcontrol reload_rules
# udevstart
Limitations
As noted above, when running under Linux, the HydraHarp software uses Libusb. However, the older version
0.1 of Libusb has an internal limitation of the block size it can handle in one transfer. Therefore, you may
observe lower USB throughput than under Windows. The throughput limitation of Libusb 0.1 can be lifted by
the combination of Libusb 1.0 and the compatibility layer Libusb-Compat that is installed by default on all recent
Linux distributions.
Another limitation is the current implementation of Wine's viewer for chm help files. You may observe some
glitches when using the online help facility.
Finally, please note that running the HydraHarp software under Linux with Wine is an experimental feature that
cannot be covered by regular product support.
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All information given in this manual is reliable to our best knowledge. However, no responsibility is assumed for possible inaccuracies or
omissions. Specifications and external appearance are subject to change without notice.
PicoQuant GmbH
Unternehmen für optoelektronische Forschung und Entwicklung
Rudower Chaussee 29 (IGZ), 12489 Berlin, Germany
Telephone: +49 / (0)30 / 6392 6929
Fax: +49 / (0)30 / 6392 6561
e-mail: info@picoquant.com
WWW: http://www.picoquant.com
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