Portable
Chlorophyll Fluorometer
PAM-2500
Handbook of Operation
2.156/08.2008
1. (preliminary) Edition: August 2008
PAM_2500_03pp.doc
© Heinz Walz GmbH, 2008
Heinz Walz GmbH • Eichenring 6 • 91090 Effeltrich • Germany
Phone +49-(0)9133/7765-0 • Telefax +49-(0)9133/5395
E-mail info@walz.com • Internet www.walz.com
CONTENTS
Contents
1 Safety Instructions ....................................................................... 7
1.1 General Safety Instructions ......................................................... 7
1.2 Special Safety Instructions .......................................................... 7
2 Introduction .................................................................................. 9
2.1 Intention of this Handbook ....................................................... 10
3 Components and Setup .............................................................. 11
3.1 Basic System Components........................................................ 11
3.2 Basic System Setup ................................................................... 11
3.3 PamWin-3 Software Installation ............................................... 13
3.3.1 USB Serial Port Setting .................................................... 14
3.3.2 Wireless Bluetooth Communication ................................. 15
3.4 Accessories................................................................................ 16
3.4.1 Field Combo (optional)..................................................... 16
3.4.1.1 Touch-Screen Ultra-Mobile Personal Computer ............ 16
3.4.1.2 External Battery 000160101314 ..................................... 16
3.4.1.3 Automatic Charger 0001X.............................................. 17
3.4.2 Additional Components .................................................... 18
3.4.2.1 Special Fiberoptics 2010-F ............................................. 18
3.4.2.2 Distance Clip 60° 2010-A............................................... 18
3.4.2.3 Leaf-Clip Holder 2030-B (optional) ............................... 20
3.4.2.4 Micro Quantum/Temp.-Sensor 2060-M (optional)......... 23
3.4.2.5 Dark Leaf Clip DLC-8 (optional) ................................... 23
4 PAM-2500 Operation................................................................. 25
4.1 PamWin-3 Help......................................................................... 25
4.2 Field Screen............................................................................... 25
4.2.1 Monitoring Graphs............................................................ 26
1
CONTENTS
4.2.2 Program and Script Control .............................................. 27
4.2.3 Alphanumerical Area........................................................ 27
4.2.3.1 Light Control .................................................................. 29
4.2.3.2 Fluorescence Data........................................................... 32
4.2.3.3 Fluorescence Ratio Parameters....................................... 33
4.2.3.4 Additional Data............................................................... 35
4.2.4 First Measurements Using the Field Screen ..................... 36
4.3 PamWin-3: Advanced Level..................................................... 38
4.3.1 General Settings................................................................ 38
4.3.1.1 Menu Bar ........................................................................ 38
4.3.1.2 Measuring Light Frequency............................................ 45
4.3.1.3 Induction Curve Parameters............................................ 45
4.3.1.4 Global Settings ............................................................... 46
4.3.1.5 PAR Sensor Control ....................................................... 47
4.3.1.6 Fo, Fo’ ............................................................................ 47
4.3.1.7 Tab Bar ........................................................................... 48
4.3.1.8 Pulses, Short Term Illumination ..................................... 48
4.3.1.9 Mode............................................................................... 50
4.3.1.10 Clock Event ............................................................... 50
4.3.1.11 New Record ............................................................... 51
4.4 SP-Analysis Mode .................................................................... 52
4.4.1 Slow Kinetics Window ..................................................... 52
4.4.1.1 Graph Icons..................................................................... 53
4.4.1.2 Graph Y, X ..................................................................... 53
4.4.1.3 Save Ft ............................................................................ 53
4.4.1.4 Display Control............................................................... 53
4.4.1.5 X-Axis Scaling/Zoom In................................................. 54
4.4.1.6 Slow Kinetics Control..................................................... 54
2
CONTENTS
4.4.2 Light Curve ....................................................................... 55
4.4.2.1 Display Control............................................................... 55
4.4.2.2 LC (Light Curve) Control ............................................... 56
4.4.2.3 Light Curve Edit ............................................................. 57
4.4.2.4 Light Curve Fit................................................................ 57
4.4.3 Report................................................................................ 58
4.4.3.1 Report Data Management ............................................... 58
4.4.3.2 Record Header and Last Line ......................................... 60
4.4.3.3 Record Columns ............................................................. 61
4.5 Fast Acquisition Mode .............................................................. 62
4.5.1 Fast Settings...................................................................... 62
4.5.1.1 Basic Settings.................................................................. 63
4.5.1.2 Menu Bar ........................................................................ 64
4.5.1.3 Trigger Settings ( I ) and ( II ) ........................................ 64
4.5.1.4 Light & Pulses ................................................................ 69
4.5.1.5 Start Fast Kinetics ........................................................... 70
4.5.2 Fast Kinetics ..................................................................... 70
4.5.2.1 Cursor Coordinates ......................................................... 71
4.5.2.2 File Names ...................................................................... 71
4.5.2.3 File Menu........................................................................ 72
4.5.2.4 Fast Kinetics ................................................................... 72
4.5.2.5 Polyphasic Fluorescence Rise......................................... 74
4.6 View Mode................................................................................ 74
4.6.1 Icon Bar............................................................................. 76
4.6.2 Sidebar .............................................................................. 76
4.6.3 Pick Data........................................................................... 76
4.7 Script Files ................................................................................ 76
5 Definitions and Equations ......................................................... 79
3
CONTENTS
5.1 Relative Fluorescence Yields.................................................... 79
5.1.1 Measurements with dark-acclimated samples................... 79
5.1.2 Measurements with light-exposed (treated) samples........ 79
5.2 Fluorescence Ratio Parameters ................................................. 80
5.3 Constant Fraction of Fo Fluorescence (C/Fo)........................... 84
5.4 Relative Electron Transfer Rate (ETR)..................................... 85
5.5 Rapid Light Curves ................................................................... 87
5.5.1 Some Papers on Rapid Light Curves ................................ 89
5.6 Literature Cited in Chapter 5 .................................................... 90
6 Some Reviews on Chlorophyll Fluorescence ........................... 93
7 Specifications .............................................................................. 95
7.1 Basic System ............................................................................. 95
7.1.1 General Design ................................................................. 95
7.1.2 Light sources..................................................................... 96
7.1.3 Special Fiberoptics 2010-F ............................................... 96
7.1.4 Distance Clip 60° 2010-A................................................. 97
7.1.5 Battery Charger MINI-PAM/L ......................................... 97
7.1.6 External Voltage Supply Cable ........................................ 97
7.1.7 System Control and Data Acquisition .............................. 97
7.1.8 Carrier Bag........................................................................ 98
7.1.9 Transport Box 2040-T ...................................................... 98
7.1.10 Minimum Computer Requirements .................................. 98
7.2 Accessories ............................................................................... 99
7.2.1 Ultra-Mobile Personal Computer for Field Research ....... 99
7.2.1.1 Computer box ................................................................. 99
7.2.1.2 Ultra-mobile touchscreen computer SAMSUNG Q1 Ultra
99
7.2.2 External battery 000160101314...................................... 100
4
CONTENTS
7.2.3 Automatic charger 000190101099.................................. 100
7.2.4 Leaf-Clip Holder 2030-B................................................ 100
7.2.5 Arabidopsis Leaf Clip 2060-B ........................................ 100
7.2.6 Dark Leaf Clip DLC-8 .................................................... 101
7.2.7 Micro Quantum/Temperature-Sensor 2060-M ............... 101
7.2.8 Suspension Cuvette KS-2500 ......................................... 101
7.2.9 Magnetic Stirrer MKS-2500 ........................................... 102
7.2.10 Compact Tripod ST-2101A ............................................ 102
8 Trouble Shooting...................................................................... 103
9 Warranty................................................................................... 105
9.1 Conditions ............................................................................... 105
9.2 Instructions.............................................................................. 106
10 Index .......................................................................................... 107
5
SAFETY INSTRUCTIONS CHAPTER 1
1 Safety Instructions
1.1 General Safety Instructions
a ) Read the safety instructions and the operating instructions
first.
b ) Pay attention to all the safety warnings.
c ) Keep the device away from water or high moisture areas.
d ) Keep the device away from dust, sand and dirt.
e ) Always ensure there is sufficient ventilation.
f ) Do not put the device anywhere near sources of heat.
g ) Connect the device only to the power source indicated in the
operating instructions or on the device.
h ) Clean the device only according to the manufacturer’s rec-
ommendations.
i ) If the device is not in use, remove the mains plug from the
socket.
j ) Ensure that no liquids or other foreign bodies can find their
way inside the device.
k ) The device should only be repaired by qualified personnel.
1.2 Special Safety Instructions
The PAM-2500 is a highly sensitive instrument which should be only
used for research purposes, as specified in this manual. Follow the
instructions of this manual in order to avoid potential harm to the
user and damage to the instrument.
The PAM-2500 can emit very strong light! In order to avoid harm to
your eyes, never look directly at the fiberoptics end, or at open light
ports at the front side of the control unit.
7
INTRODUCTION CHAPTER 2
2 Introduction
The PAM-2500 Portable Chlorophyll Fluorometer is the follow-up
model of the well-known PAM-2000/2100 instruments which were
introduced in the 1990s as the first portable PAM fluorometers and
since then have been successfully applied worldwide by numerous
scientists. In the development of the PAM-2500, particular care was
taken to maintain all properties appreciated by PAM-2000/2100 users
and, at the same time, to take account of the recent technical progress.
Essentially, the hardware and optical system are thoroughly modernized. Also, while continuing basic elements of the graphical user interface, instrument operation is based on the newly-developed PamWin-3 software. The program permits operation under Windows operating systems on normal personal computers, but also on ultra mobile touch screen computers.
Major points of progress of the PAM-2500 with respect to its predecessors are:
- Use of LEDs (light emitting diodes) for all internal light
sources including Saturation Pulses and Actinic Light.
- Blue and Red Actinic internal light sources.
- Single turn-over and multiple turn-over saturating flashes.
- Time resolution down to 10 µs.
- Easily updateable firmware.
- Optional touch-screen operation by an ultra-mobile com-
puter.
9
CHAPTER 2 INTRODUCTION
2.1 Intention of this Handbook
The Portable Fluorometer PAM-2500 displays a high degree of flexibility in measuring and analyzing fluorescence. This does not mean,
however, that all features of this multifunctional instrument must be
understood before measurements can be started. Actually, due to the
"intelligent" central control of all functions by the special PamWin-3
software, serious operational mistakes harming the instrument are
highly unlikely. Also, at first there is no need to care about the numerous settings of instrument parameters, because these are preadjusted for standard measurements. Hence, even the inexperienced
user can start measuring with a minimum of background knowledge,
and will be gradually guided to deeper understanding and more profound applications.
This handbook tries to cover all of the numerous features and applications of the PAM-2500 Fluorometer, some of which probably are
not of immediate interest to many users, but probably will become
relevant, as new questions arise on the basis of the results obtained.
The best way to become acquainted with all features of the
PAM-2500 Fluorometer is to read this handbook section by section,
trying out all described functions. On the other hand, in order to get a
quick start it will suffice to read Chapter 4.2.
10
COMPONENTS & SETUP CHAPTER 3
3 Components and Setup
3.1 Basic System Components
The basic system consists of the following components:
a ) PAM-2500 Control Unit
b ) Special Fiberoptics 2010-F
c ) Distance Clip 60° 2010-A
d ) Battery Charger MINI-PAM/L
e ) MINI-PAM/AK cable
f ) Special USB cable PAM-2500/K1
g ) Fluorescence Standard Foil
h ) Spare Fuse
i ) Carrier Bag
j ) Transport Box 2040-T
k ) Software. PamWin-3 System Control and Data Acquisition
3.2 Basic System Setup
Note: Great caution should be exercised to prevent any dirt or for-
eign matter from entering the ports for the fiberoptics pins.
To set up the PAM-2500 fluorometer:
- Connect fiberoptics to the control unit using the three-pin op-
tical connector at the front of the unit (Fig. 3.1).
11
CHAPTER 3 COMPONENTS & SETUP
Note: Do not force a plug into the wrong socket. Orientate each plug
so that the red dot on the plug coincides with the red dot of the
socket. Do not try to disconnect a plug by pulling at the cable.
Disconnect plug by pulling at the rippled metal part of the plug.
Figure 3.1: PAM-2500 Control Unit. Front. (AL: Red Actinic Light, SP:
Saturation Pulse, ML: Measuring Light, FR: Far-Red light, BL: Blue Actinic Light, Det.: Detector)
12
COMPONENTS & SETUP CHAPTER 3
- Connect the battery charger cable to the <EXT.DC> socket
(Fig. 3.1 A), and to line power: thereafter, a green
<CHARGE> LED (Fig. 3.1 A) indicates that the internal battery is charged but yellow LED light signifies that the battery
is currently charged. Please note that an external 12 V battery
cannot recharge the internal battery; but it can provide power
(via the MINI-PAM/AK cable) when the internal battery is
empty.
- Connect computer and control unit (USB socket, Fig. 3.1 A)
by the PAM-2500 special USB cable.
- Switch on PAM-2500 using the toggle switch labeled
POWER ON (Fig. 3.1 A). Flashing of the <Status LED>
(Fig. 3.1 A) indicates that the system is ready for communications. The LED will shine continuously once communication with the computer has been established. Constant light
from the <Status LED> in the absence of computer communication indicates malfunction of the PAM-2500. Usually,
switching power OFF and, after a couple of seconds, ON
again will restore normal function.
3.3 PamWin-3 Software Installation
Depending on the type of CD-ROM delivered with the PAM-2500
Chlorophyll Fluorometer, you have to start installation with a) or b).
a) Your <Software & Manuals CD-ROM> contains only a setup
file (e. g. <PamWin_setup.exe>), and the present handbook
in PDF file format.
- Close other programs before installation of the PamWin_3
software.
13
CHAPTER 3 COMPONENTS & SETUP
- Double click on the setup file and follow instructions. The
setup routine will create in the <C:> root directory the folder
<PamWin_3> containing the PamWin-3 main program and
accessory files. Further, an icon representing a shortcut to
PamWin-3 is created on the computer desktop.
b) Your <Software & Manuals CD-ROM> contains the com-
plete collection of the Walz Software & Manuals.
- The CD automatically starts the default internet browser of
your computer. (If automatic browser start fails, double-click
on <index.html> in the root directory of the <Software &
Manuals> CD-ROM.)
- Choose <Fluorescence Products> → <PAM-2500> → <PC
software PamWin-3>.
- Close other programs before installation of the PamWin_3
software.
- Click on <PamWin-3> to start software installation as de-
scribed above.
3.3.1 USB Serial Port Setting
For serial communication via a serial USB port, a driver software is
installed during the PamWin-3 setup procedure. The successful
driver installation will be confirmed by the software. At first start of
the PamWin-3 software, the program checks the setting of the “latency timer” which needs to be adjusted to 1 ms to ensure high rates
of data transfer.
The latency timer is set to 1 ms on the port (COM & LPT) properties
page which is accessed via the device manager. For Windows XP
computers proceed as described below (the procedure is similar for
the Windows Vista operating system):
14
COMPONENTS & SETUP CHAPTER 3
- Make sure that the PAM-2500 is switched on and connected
by the USB cable to the computer
- Open <Windows Start Menu>
- Select <Settings>
- Select <Control Panel>
- Select <System> and left-click the <Hardware> tab
- Left-click the <Device Manager> button
- Open folder <Ports & LPT>
- Select <USB Serial Port COM#> (double click with left
mouse key).
- Select <Port Settings> tab with left mouse key
- Left-click the <Advanced> button
- Set latency timer to 1 ms
- Close by clicking <OK> in the <Advanced Settings> win-
dow, and the <USB properties> window
3.3.2 Wireless Bluetooth Communication
- Please note that Bluetooth data transfer is too slow for the
<Fast Kinetics> mode of PamWin-3. The <Fast Kinetics>
mode is not available when in Bluetooth communication is
active.
- Make sure that the PAM-2500 is switched on and NOT con-
nected via USB cable to a computer.
- Open <My Bluetooth Places> in the <Start> menu or on the
computer desktop.
- Start by double-click <Find Bluetooth devices>
15
CHAPTER 3 COMPONENTS & SETUP
- Now, the device <PAM 2500 SNR XXXX> should be in the
list of Bluetooth devices (the four “X” stand for the four digit
serial number). If the PAM 2500 is not listed, change positions of PAM 2500 relative to computer. (Hint: the Bluetooth
output antenna is located at the rear top face of the PAM
2500 instrument.)
- Double click on <PAM 2500 SNR XXXX> and, subse-
quently on <SerialPort on PAM 2500 SNR XXXX>
- Now you’ll be prompted to enter a <Bluetooth security code>
which is for all PAM 2500 chlorophyll fluorometers the
number:
2500
- The Bluetooth connection is now ready for use.
3.4 Accessories
See Chapter 7 for specifications of accessories.
3.4.1 Field Combo (optional)
The add-ons summarized as <Field Combo> convert the PAM-2500
chlorophyll fluorometer into a highly mobile field station, equipped
with the options that a normal PC offers and working for hours independent of line current.
3.4.1.1 Touch-Screen Ultra-Mobile Personal Computer
3.4.1.2 External Battery 000160101314
16
COMPONENTS & SETUP CHAPTER 3
3.4.1.3 Automatic Charger 0001X
To assemble the <Field Combo> proceed as shown in Fig. 3.2 and
described subsequently:
- Piece together <External Battery> and rectangular rubber
foam filler (Fig. 3.2 A) and place in PAM-2500 carrier bag as
shown in (Fig. 3.2 B and C).
- Slide-in computer box below elastic belt (Fig. 3.2 C and D).
Note that the computer box is designed to accept the highcapacity battery of the Ultra-Mobile Personal Computer.
- Insert Ultra-Mobile Personal Computer (Fig. 3.2 E).
Figure 3.2: PAM-2500 Field Setup
17
CHAPTER 3 COMPONENTS & SETUP
3.4.2 Additional Components
3.4.2.1 Special Fiberoptics 2010-F
The Special Fiberoptics 2010-F are connected to the front side of the
PAM-2500 control unit with the help of a special plug that resembles
an electrical connector. There are three "fiber pins" with different optical cross-sections, which fit into the corresponding holes at the front
side of the PAM-2500 housing, where they interface the various light
sources and the photodiode detector. Within the "interface plug" the
three fiber branches are joint to a common fiber bundle and randomized via a 100 cm mixing pathway.
Note: The fiberoptics should be handled with care. Excessive bend-
ing, in particular close to the connector plug, should be
avoided, as it would lead to fiber breakage resulting in a loss
of signal amplitude. The fibers are protected by a steel-spiral
and plastic mantle, which provide a natural resistance to
strong bending.
3.4.2.2 Distance Clip 60° 2010-A
A Distance Clip is provided with the fiberoptics for convenient positioning of the fiberoptics end-piece relative to the sample. The axis of
the end-piece is positioned at a 60° angle relative to the sample plane
(angle of incidence, 30°). Minimum shading of the sample is
achieved when the fiberoptics points towards the sample from the
side opposite to incident light. Two spacer rings may be used to define fixed distances.
The sample may be placed either below the hole (e. g. thick leaves,
lichens and mosses) or, preferentially with normal leaves, above the
18
COMPONENTS & SETUP CHAPTER 3
hole (compare Fig. 3.3). In the latter case, the leaf can be held between the folded part of the clip.
Figure 3.3: Distance Clip (to position leaf with respect to fiberoptics)
The distance between fiberoptics exit plane and sample has considerable influence on signal amplitude and effective light intensities (Fig.
3.4). Clearly, with a 60° angle between sample plane and fiberoptics,
the distance between fiber optics tip and leaf surface varies, and,
hence, the leaf surface is exposed to slightly heterogeneous light intensities. A much more pronounced intensity gradient exists inside
the leaf due to shading by the top chloroplast layers. In essence, the
measured signal will be dominated by that part of the leaf which re-
Figure 3.4: Relationship between signal amplitude/light intensity and
distance between fiberoptics exit plane and sample
19
CHAPTER 3 COMPONENTS & SETUP
ceives maximal intensity, as this also is most strongly excited by the
Measuring Light and emits most of the fluorescence which is received by the fiberoptics.
3.4.2.3 Leaf-Clip Holder 2030-B (optional)
The Leaf-Clip Holder 2030-B is connected to the <LEAF CLIP>
socket (Fig. 3.1 A) for recording of PAR and temperature in parallel
with chlorophyll fluorescence.
The Leaf-Clip Holder 2030-B is almost indispensable for field investigations, when ambient light and temperature conditions may vary
considerably. It substitutes for the standard "Distance Clip" as a device for defined positioning of the fiberoptics relative to the leaf
plane. It features special micro-quantum and temperature sensors, the
readings of which are transferred to the PAM-2500 with every Saturation Pulse measurement.
Figure 3.5: Leaf-Clip Holder 2030-B with Fiberoptics 2010- F.
20
COMPONENTS & SETUP CHAPTER 3
In this holder, the leaf is resting on a Perspex tube with widened
crest, which can be vertically adjusted, to account for different leaf
thicknesses. The fiberoptics axis forms a 60° angle with the leaf
plane. Optionally, a 90° fiberoptics adapter (2030-B90) is available.
The distance between fiberoptics and leaf can be varied. Standard
distances are defined by spacer rings. The illuminated leaf area is
limited by a steel ring with 10 mm ∅ opening.
At the bottom of the Leaf-Clip Holder 2030-B, a tripod mounting
thread is provided. Mounting the device on a tripod (e. g. Compact
Tripod ST-2101) facilitates long term measurements with the same
plant.
The handle of the Leaf-Clip Holder 2030-B features a red pushbutton for remote control of the PAM-2500. Pressing this button initiated a Saturation Pulse.
Micro-Quantum-Sensor
A micro quantum sensor is integrated into the Leaf-Clip Holder
2030-B to monitor the photosynthetic active radiation (PAR) to
which the sample is exposed. The micro-quantum-sensor measures
incident PAR in µmol quanta/(m
2
·s), i.e. in units of flux density.
Hence, the measured parameter PAR is identical to PPFD (photosynthetic photon flux density).
Essential optoelectronic elements of this micro-quantum-sensor are:
- a 1.5 mm ∅ diffusing disk
- a 0.5 mm diameter fiber guiding the scattered light to the de-
tector
- a filter combination selecting the photosynthetic active wave-
length range between 380 and 710 nm
- a blue-enhanced silicon photodiode
21
CHAPTER 3 COMPONENTS & SETUP
The sensor is factory calibrated against a standard lamp. Radiation of
the standard lamp approached the sensor surface perpendicularly, that
is at an angle of incidence of 0°. Therefore, the 2030-B sensor measures with adequate accuracy collimated light incident at low angles,
but also completely diffuse radiation. The angular response of the
sensor, however, deviates from the ideal cosine behavior: to be exact,
the sensor overvalues collimated light impinging at greater angles of
incidence. Therefore, when the PAM-2500 internal Actinic Light
sources are applied via the fiberoptics (Fig. 3.5), e.g. for recording of
light response curves, the sensor should be switched off via software
(see Advanced Mode, General Settings, Section 4.3.1) and the PAR
should be derived from previously defined PAR-lists (see Section
4.3.3.1).
The stability of calibration depends on keeping the diffuser clean. It
is advisable to check calibration regularly by comparison with a standard quantum sensor. Any deviation can be corrected by entering a
recalibration factor in the PamWin-3 program (see Section 4.3.1.1).
A substantial increase of the calibration factor from its original value
of 1.000 indicates dirt-deposition on the diffuser, which may be reversed by gentle cleaning using a cotton tip applicator, moistened
with some ethanol.
Thermocouple Monitoring Leaf Temperature
A NiCr-Ni thermocouple is mounted in the Perspex tube on which
the investigated leaf area is resting. Its tip is forming a loop that gently presses against the lower surface of the leaf. In this way there is
effective temperature equilibration and the thermocouple is protected
from direct sun radiation. The reference couple is located on the circuit board, in close proximity to the thermovoltage amplifier, enclosed in the bottom part of the holder. The relationship between
thermovoltage and temperature is almost linear. With decreasing
temperatures there is a small decline of ΔV/°C. Calibration was per-
22
COMPONENTS & SETUP CHAPTER 3
formed at 25 °C. At 0 °C or –15 °C the deviation amounts to 0.5 or
0.8 °C, respectively. An offset value can be entered in the PamWin-3
program (see Section 4.3.1.1).
The temperature, as well as the PAR data, is automatically stored in
the Report-file after every saturation pulse, together with the on-line
calculated quenching parameters.
3.4.2.4 Micro Quantum/Temp.-Sensor 2060-M (optional)
The Micro Quantum/Temp.-Sensor 2060-M essentially displays the
same features as outlined above for the Leaf-Clip Holder 2030-B, except that the micro-sensors of PAR and temperature are not mounted
in a leaf-clip. This device is rather designed for experiments with objects which are not leaf-shaped, like crustose lichens and cushions of
moss. The two miniature sensors can be attached to the site where
fluorescence is monitored without interfering with the actual measurement. A defined position with respect to the object and the fiberoptics exit plane can be achieved with the help of a special holder, in
analogy to the "Distance Clip" (see above).
It should be pointed out that the sensitivity of the micro quantum sensor is affected by bending the relatively long, flexible light guide that
bridges the distance between the small diffusing disk at the object
and the detector in the metal housing. Therefore, this device cannot
substitute for a reliable quantum sensor. Recalibration is recommended after bringing the sensor and the metal housing into a fixed
position with respect to the object.
3.4.2.5 Dark Leaf Clip DLC-8 (optional)
The Dark Leaf Clip DLC-8 weighs approx. 4 g and, hence, can be attached to most types of leaves without any detrimental effects. It is
equipped with a miniature sliding shutter which prevents light access
23
CHAPTER 3 COMPONENTS & SETUP
to the leaf during a dark-adaptation period. This shutter is opened for
the actual measurement only, when exposure to external light is prevented by the fiberoptics. Proper dark-acclimation is essential for determination of the maximal quantum yield Fv/Fm and for recording
of dark-light induction kinetics.
Figure 3.6: Dark Leaf Clip
Using the Dark Leaf Clip DLC-8, the fiberoptics is positioned at right
angle with respect to the leaf surface at the relatively short distance
of 7 mm. As a consequence, signal amplitude is distinctly higher than
when the Leaf-Clip Holder 2030-B with 60° fiberoptics angle is used.
In order to avoid signal saturation, the settings of Measuring Light
Intensity and Gain have to be correspondingly lowered with respect
to the standard settings (see Section 4.2).
When the shutter is still closed and the Measuring Light is on, an artifactual Ft signal is observed. This signal is due to a small fraction of
the Measuring Light which is reflected from the closed shutter to the
photodetector. However, this background signal is of no concern as
the reflection is much smaller when the shutter is opened and the
Measuring Light hits the strongly absorbing leaf instead of the metal
surface of the shutter that acts like a mirror.
24
PAMWIN-3 FIELD SCREEN CHAPTER 4
4 PAM-2500 Operation
4.1 PamWin-3 Help
All program levels of the PamWin-3 software offer online Help-texts
with information on essentially all active user surface elements. To
access the online Help-texts:
- Move mouse cursor on the window element of interest until a
small tag (“Tooltip”) appears.
- A “Tooltip” remains visible for 2.5 seconds. During this inter-
val, pressing the <@> or <F1> key results in the display of the
relevant Help-text.
For getting acquainted with the instrument and its software, it is
strongly recommended to make frequent use of this function.
4.2 Field Screen
The <Field Screen> of the PamWin-3 software has been developed
for outdoor operation of the PAM-2500 fluorometer where ease and
simplicity of instrument control is important. Conveniently, the elements of the <Field Screen> are accessible via the display of a touch
screen PC. The <Field Screen> is divided into three areas containing
(1) alphanumeric fields, (2) program and Script control, and (3)
monitoring graphs, respectively (see Fig. 4.1).
The alphanumeric area contains the switches for light control and
data derived from Saturation Pulse analysis. It also includes the
Zoom In button: the <Zoom In> command results in the screen
display of the alphanumeric area.
25
CHAPTER 4 PAMWIN-3 FIELD SCREEN
4.2.1 Monitoring Graphs
The <Field Screen> provides 2 monitoring graphs (Fig. 4.1): the left
graph records slow fluorescence changes, and also depicts Fm and
Fm’ values as small crosses. Generally, only those Saturation Pulse
analyses which result in graphical display of Fm or Fm’ values are
entered in the Report file - Saturation Pulses analyses carried out
with stopped monitoring screen are not reported. To view the Report
file click the Report icon. Editing of the Report is restricted to the
<Advanced> level of the PamWin-3 program.
Monitoring of slow kinetics begins automatically with PamWin-3
program start. The slow kinetics monitoring graph is restarted by an
<Fo, Fm> determination via Fv/Fm (see below) or via the
Stop / Start button. An Fv/Fm determination automatically adapts
the Y axis range of both monitoring screens to the signal amplitude.
The actual signal size (in Volts) can be read from the Y-axes. In both
Figure 4.1: Field Screen Overview.
26
PAMWIN-3 FIELD SCREEN CHAPTER 4
screens, the Fo and Fm levels are displayed as yellow and red dashed
line, respectively. Always, the time axis of the slow kinetics monitoring screen covers an interval of 5 minutes which is automatically
shifted to the left for monitoring times > 5 minutes.
The right monitoring graph displays the fast fluorescence kinetics induced by last Saturation Pulse over a time interval of 1.6 seconds. In
addition to the Fo and Fm lines, the determined Fm’ level is depicted
as a dashed black line.
4.2.2 Program and Script Control
The Program and Script Control section (Fig. 4.1) includes buttons to
quit the program ( Exit PamWin ), to access to the full range of
PamWin-3 capabilities ( Advanced ), and to load Script files which
carry out automatically preprogrammed experimental protocols
(Script: Load and Run ).
4.2.3 Alphanumerical Area
The elements of the alphanumerical area are divided into four groups:
(1) light control, (2) primary fluorescence data, (3) fluorescence ratio
parameters, and (4) additional data like PAR and temperature (Fig.
4.2).
Group 1 (Light Control, see Fig. 4.2) includes “status” and “adjust-
ment” buttons. A status button either turns on or off a function (e.g.
the Measuring Light) or it changes the light color (e.g. red to blue
Actinic Light). To change a status, click on the status button with the
left mouse key. Status buttons with on/off-switching function display
green background when the associated function is active. The status
buttons controlling light color, display the selected type of light as
text information.
27
CHAPTER 4 PAMWIN-3 FIELD SCREEN
Adjustment buttons provide access to settings of parameters (e.g.
light intensity or clock interval). Settings can be adjusted in different
ways: (1) single click with left mouse key on the button and turn the
mouse wheel, (2) double-click with left mouse key on the button and
enter the value, or (3), when a ultra-mobile computer is used, tap on
the button and modify the value using the up/down arrows.
All parameter fields of group 2 to 4 display information related to
the current Saturation Pulse measurement. The “Fluorescence Ratio
Parameters” (group 3) also contain two fields to trigger Saturation
Pulse analyses: the Fv/Fm and Y(II) buttons (see 4.2.3.2). In
comparison to the latter, the Sat. Pulse button of group 1 ele-
ments triggers a Saturation Pulse without Saturation Pulse Analysis.
Figure 4.2: Alphanumerical Area of Field Screen.
28
PAMWIN-3 FIELD SCREEN CHAPTER 4
4.2.3.1 Light Control
First Column
Meas Light The <Meas Light> button is the on-off switch of
Measuring Light.
Int. Measuring Light intensity is adjusted by changing the level in
the <Int.> field. 20 different intensity levels are available. The Measuring Light intensity varies nearly linearly with the level number.
While at low frequency the actinic effect of the Measuring Light can
be neglected, its integrated intensity can be appreciable at high frequency. When PAR is not recorded by an external sensor, the Measuring Light intensity is derived from the currently active internal
light list (see 4.3.1.1 AL Current/PAR Lists).
MF-H Clicking on the <MF-H> button switches between low and
high Measuring Light frequency. Grey background color indicates
low frequency (e.g., for Fo determination) and green background
color indicates high frequency (e.g., during actinic illumination).
Measuring Light frequencies can be modified at the Advanced Level
of WinControl-3.
Gain Ten different levels for electronic signal amplification are
provided: at Gain 10 the signal is amplified 7-fold compared to
Gain 1.
Damp Ten different levels of electronic signal damping are pro-
vided ranging from damping switched off (setting 1, t(1/2) = 10 µsec)
to maximum damping (setting 8, t(1/2) = 4 ms). Note that at low
Measuring Light frequencies, the response time is determined by the
current sampling rate and, hence, much lower than the damping levels specified above.
29
CHAPTER 4 PAMWIN-3 FIELD SCREEN
Second Column
Act. Light Clicking on this button switches Actinic Light on or
off.
Int. Actinic light intensity is adjusted as described above. For dif-
ferent optical geometries, the PAR produced by the red and blue internal light sources are provided as internal light lists which are
available in the Options menu at the Advanced Level (see 4.3.1.1 AL
Current/PAR Lists).
s: The time interval in seconds of Actinic illumination is defined
via the <s:> button. For <s: 0>, both, turning on and off the Actinic
Light, is carried out manually by clicking on the <Act. Light> button.
Red / Blue The button toggles between red and blue actinic illu-
mination.
Auto MF-H When the <Auto MF-H> function is activated, switch-
ing on Actinic Light automatically increases the Measuring Light
frequency from low to high. The status of <Auto MF-H> does not affect the Measuring Light frequency during Saturation Pulses which is
always 100 kHz.
Third Column
Sat. Pulse Clicking on the <Sat. Pulse> button releases a Satura-
tion Pulse without carrying out a Saturation Pulse Analysis.
Int. This intensity button allows adjusting the intensity of Satura-
tion Pulses. Typical PAR values for settings 1 and 20 are 910 and
16500 µmol photons/(m
2
·s), respectively. Setting number and PAR
values are quasi-linearly related. (PAR measured by MQS-B Quantum Sensor and a Universal Light Meter (ULM, Walz) at standard
distance and 60° optical geometry of a 2030-B Leaf-Clip Holder.)
30
PAMWIN-3 FIELD SCREEN CHAPTER 4
0.1 s: Using this button permits to define the duration of a Satura-
tion Pulse in tenths of a second.
Clock Activation of <Clock> initiates repetitive delivery of satu-
rating flashes.
s: The button allows to define the time interval (in seconds) be-
tween 2 Saturation Pulses in a pulse sequence (see above).
Fourth Column
PS I Light Clicking on the <PS I Light> button switches on the
alternative light source. When Actinic Light is set to red, <PS I
Light> is either far-red radiation (LED emission peak at 740 nm) or
blue radiation with maximum emission at 460 nm. The far-red preferably excites PS I in plants and many eukaryotic algae but the blue
is used to specifically excite PS I in cyanobacteria. Of course, with
red as the Actinic Light, blue <PS I light> can be used as a second
Actinic Light source with non-cyanobacterial algae and plants.
lnt. Intensity adjustment of <PS I Light> occurs as described for
other light sources. Typical blue light intensities are reported together
with red Actinic Light on the <Advanced> level of PamWin-3 (see
Section 4.3.1.1 AL Current/PAR Lists). We do not report far-red
light intensities which would be misleading because only a small part
of the far-red is absorbed by PS I and the effectiveness of far-red to
excite PS I may vary markedly between species.
s: The time interval in seconds of <PS I light> is defined via the
<s:> button. For <s: 0>, both, turning on and off the PS I light, is carried out manually by clicking on the <PS I Light> button.
FR / Blue The button toggles between far and blue illumination
as the second light source. With blue as the second light source, the
Actinic Light is always red.
31
CHAPTER 4 PAMWIN-3 FIELD SCREEN
4.2.3.2 Fluorescence Data
Dark-acclimated sample
With a dark-acclimated sample, clicking on Fv/Fm records two
types of Saturation Pulse data (see “Fluor.Data” in Fig. 4.2):
Fo Basic chlorophyll fluorescence yield recorded with low Measuring Light intensities.
Fm Maximal chlorophyll fluorescence yield when photosystem II
reaction centers are closed by a Saturation Pulse.
Light-exposed sample
In response to a click on the Y(II) button, three types of Saturation
Pulse data are recorded with a light-exposed sample: the Fo’, the
Fm’, and the F which is the value for Ft shortly before a Saturation
Pulse:
Fo’ Minimum chlorophyll fluorescence yield in the state of open
photosystem II reaction centers. The Fo’ is measured in the presence
of far-red illumination with Actinic Light switched off. To activate
the Fo’ measuring mode, proceed to <Advanced Level> and <General Settings>. When the Fo’ mode is inactive, the <Fo’> field displays the value of 0. In the presence of non-photochemical quenching, the Fo’ is lowered with respect to Fo.
Fm’ Maximal chlorophyll fluorescence yield when photosystem II
reaction centers are closed by a strong light pulse. The Fm’ is lowered with respect to Fm by non-photochemical quenching.
Ft The Ft denotes the continuously recorded fluorescence. The
value of Ft measured shortly before a Saturation Pulse with lightexposed samples is denoted “F”. Unlike the previous fluorescence
levels, the value of F is not displayed permanently.
32
PAMWIN-3 FIELD SCREEN CHAPTER 4
4.2.3.3 Fluorescence Ratio Parameters
PS II yield
Two fluorescence ratio parameters are calculated to estimate the efficiency of photosystem II to use excitation energy for photochemistry
(see Fig. 4.2, PS II Yield):
Fv/Fm = (Fm-Fo)/Fm = Y(II)max; maximum photochemical quan-
tum yield of photosystem II, normally observed after darkacclimation (cf. 4.2.3.2). Consequently,
Y(II) = (Fm’-F)/Fm’; effective photochemical quantum yield of
photosystem II. The Y(II) is lowered with respect to
Y(II)max by non-photochemical down-regulation and reaction center closure.
ETR, Y(NPQ), Y(NO), Fluoresc. Quenching
In addition to PS II yield data, seven fluorescence ratio parameters
and the relative electron transport rate (ETR) are evaluated. These
data are shown against variable background colors (cf. Fig. 4.2). The
background colors match the symbol colors used to graph data at the
<Advanced> level of PamWin-3. For all measured and calculated
data, definitions are provided in Chapter 5: <Definitions and Equations>.
Only four of the eight parameters can be displayed at a time. Therefore, for each of the 4 display panels (see: ETR, Y(NPQ), Y(NO),
Fluoresc. Quenching. Fig. 4.2), one parameter can be selected from a
list which appears after a left-click on a parameter field.
When Fo’ is measured with a light-exposed sample during post-pulse
illumination by far-red light, two of the eight parameters (qP and qL,
see Chapter 5) can be calculated without fluorescence data from the
dark-acclimated sample, that is without Fo and Fm. Calculations of
33
CHAPTER 4 PAMWIN-3 FIELD SCREEN
qP and qL, however, require Fo and Fm measurements when the Fo’
is derived from Fo, Fm and Fm’ data according to Oxborough and
Baker (1997) (see Chapter 5). The eight parameters are (see Chapter
5 for definitions):
ETR Electron transport rate in µmol electrons/(m
2
·s) derived from
Y(II) and PAR.
˜Fo’ Calculated minimum chlorophyll fluorescence yield in the
state of open reaction centers.
qL Coefficient of photochemical fluorescence quenching assuming
that all reaction centers share a common light-harvesting antenna
(lake model). In comparison, the qP is based on a model of separate
photosynthetic units (puddle model).
Y(NO) Quantum yield of non-photochemical energy conversion in
PS II other than that caused by down-regulation of the lightharvesting function.
YNPQ Quantum yield of non-photochemical energy conversion in
PS II due to down-regulation of the light-harvesting function.
Note: Y(II)+ Y(NPQ)+ Y(NO)=1 (complementary quantum yields)
NPQ Non-photochemical fluorescence quenching: quantification of
non-photochemical quenching alternative to qN calculations. The extent of NPQ has been suggested to be associated with the number of
quenching centers in the light-harvesting antenna.
qN Coefficient of non-photochemical fluorescence quenching,
ranging from 0 (in the dark-acclimated state) to 1.
qP Coefficient of photochemical fluorescence quenching, ranging
from 0 (upon application of a Saturation Pulse) to 1 (in the darkacclimated state); based on a model of separate photosynthetic units
(puddle model).
34
PAMWIN-3 FIELD SCREEN CHAPTER 4
4.2.3.4 Additional Data
Tmp With a Leaf-Clip Holder 2030-B connected, the temperature
in °C of the lower leaf side is displayed.
PAR With an external PAR sensor connected, the measured photo-
synthetic active radiation in µmol photons/(m2 · s) is displayed. Read-
ing of data from the PAR sensor can be switched off on the <General
Settings> window in the Advanced Mode of PamWin-3. Without external PAR measurement, the PAR is derived from the currently active internal PAR list (see Section 4.3.1.1 AL Current/PAR Lists).
Note that the PAR sensor of the Leaf-Clip Holder 2030-B does not
give correct readings for Actinic Light applied via the fiberoptics,
unless properly calibrated for this purpose (cf. Section 3.4.2.3). It is
therefore recommended to derive PAR values from the internal PAR
lists when an Actinic Light source of the PAM-2500 is used.
Volt Battery voltage. A completely charged battery shows voltages
up to 13.7 Volts. At Voltages below 10.5 Volts, the PAM-2500 operates unreliable, particularly during delivery of Saturation Pulses
which require high current flow.
- Note: to prevent deep discharge of battery, the PAM-2500 shuts
off when the battery voltage drops below 9.4 Volts.
- Note: at battery voltages below 11.8 Volts, the PAM-2500 can-
not be switched on.
No The <NO> displays the number of Yield-determinations by the
Saturation Pulse method in the current Record. Counting is reset by
the <Start/Stop> button of the field screen.
35
CHAPTER 4 PAMWIN-3 FIELD SCREEN
4.2.4 First Measurements Using the Field Screen
This section introduces to basic chlorophyll fluorescence measurements using the Field Screen.
Firstly, make sure that the Measuring Light is switched on. As long
as there is no chlorophyll containing object, the Ft parameter field
shows values close to 0. When you approach a leaf with the fiberoptics, fluorescence is excited and guided via the fiberoptics to the detector system. Depending on the distance, more or less Ft will be
measured. For reproducible measurements the distance between fiberoptics exit and leaf should be constant. For this purpose, leaf and
fiber end needs to be fixed, e.g. using the small Distance Clip 60°
2010-A or the Leaf-Clip Holder 2030-B.
Information on photosynthesis is obtained when the yields of fluorescence in the presence of different illumination conditions are compared. For this purpose the PAM-2500 contains various light sources.
When you click the Act. Light key, you will see that the leaf is il-
luminated by a relatively strong red light. At the same time the value
of Ft quickly rises and then slowly decays again. Here you witness
the so-called "Kautsky-effect". Clicking the Act. Light key again
turns off the Actinic red light resulting in an Ft decrease.
With far-red radiation selected, clicking on the PS I Light key im-
mediately after shutting-off the Actinic Light, will start a far-red illumination for the set time interval (typically 3 s). In the presence of
far-red light, the decline of Ft is speeds up, with Ft approaching the
fluorescence level before Actinic Light exposure. The effect of farred light on fluorescence yield can be explained in the framework of
the so-called Z-scheme of photosynthesis and by the theory of fluorescence quenching: the far-red is preferably absorbed by PS I which,
in the absence of actinic illumination, withdraws electrons from the
36
PAMWIN-3 FIELD SCREEN CHAPTER 4
intersystem electron chain and, thus, quickly opens PS II reaction
centers.
The minimal fluorescence yield, called Fo, is observed when all PS II
reaction centers are open, which is the case after dark-acclimation.
The maximal fluorescence yield, called Fm, is observed when all
PS II centers are closed. Full closure of reaction centers and consequent Fm-determination is achieved by a Saturation Pulse that is applied by clicking the Fv/Fm key. Actually, this command deter-
mines Fo and Fm quickly one after the other. At the same time, the
value of Fv/Fm is calculated and entered into the Fv/Fm field. The
latter parameter corresponds to the ratio (Fm-Fo)/Fm, which gives
information on the photochemical quantum yield of open PS II reaction centers. With a healthy and dark-adapted leaf, Fm is about five
times higher than Fo, and, hence, Fv/Fm amounts to approx. 0.8.
Saturation Pulses are also triggered via the Y(II) key. Then, a new
value is entered in the Y(II) field. As long as the Actinic Light is off,
these values will be very close to the Fv/Fm value. However, as soon
as actinic illumination is started, you will see that the values of Y(II)
first decrease, then rise again, and eventually assume a constant value
that is characteristic for the photosynthetic performance of the given
leaf sample.
The Y(II)-determination can be repetitively triggered using the
Clock key: the Saturation Pulses now will be applied at time inter-
vals defined in the <s:>-time field below the Clock key. The fluo-
rescence data measured during Saturation Pulse events are automatically stored in the Report file which can be accessed via the
Report key.
37
CHAPTER 4 PAMWIN-3 ADVANCED LEVEL
4.3 PamWin-3: Advanced Level
The <Advanced> level of the PamWin-3 software includes Saturation
Pulse analysis, fluorescence kinetics (ranging from fast changes in
the µsec domain to slow changes over many seconds, minutes or
even hours), and a wide range of graphical and analytical features.
The <General Settings> screen is the initial user interface of the
<Advanced> level. Under <General Settings>, the mode of operation
of the PAM-2500 is selected (either <SP-Analysis> or <Fast Acquisition>) and the PAM-2500 settings are adjusted. The icons and display fields on the right and below the <General Settings> window are
retained when switching to other windows of the PamWin-3 software
are selected. The details of PAM-2500 operation on the Advanced
level will be given subsequently.
4.3.1 General Settings
Figure 4.3 shows the <General Settings> window. Parameter fields
showing the same information as already outlined for the <Field
Screen>, or buttons having similar function as corresponding buttons
on the <Field Screen>, are grayed out and will not be considered in
the following sub-sections. (Note that the selection of 4 out of 8 fluorescence parameters for numerical display works as described previously: see 4.2.3.3).
4.3.1.1 Menu Bar
The menu bar of the <General Settings> window (see Fig. 4.3) contains 6 menus. The list of menu items may vary if you switch from
the <General Settings> window to another PamWin-3 window. To
access a menu item, click sequentially on menu title and item. Also,
menu items can be accessed by Windows shortcuts: press down and
38
PAMWIN-3 ADVANCED LEVEL CHAPTER 4
hold the <Alt> key, and sequentially type the first (underlined) letter
of the menu title and the first (underlined) letter of the menu item.
Underlining itself is switched on and off by the Alt key.
File
The file menu includes items for loading and execution of PamWin-3
Script files which are used to automatically perform measuring routines. Also, printer configurations and program closure can be carried
out using the <File> menu.
Figure 4.3: General Settings.
39
CHAPTER 4 PAMWIN-3 ADVANCED LEVEL
Window
The <Window> menu is equivalent to the <Tab Bar> (see Fig. 4.3).
The <Window> menu links to the 4 available windows in the <SPAnalysis> mode; in the <Fast Acquisition> mode, the menu includes
the 3 windows available when <Fast Acquisition> is carried out.
Options
L Curve Fit Parameters
The menu item <Light Curve Fit Parameters> opens a window, on
which 4 parameters are displayed that were derived by fitting a theoretical Light Response Curve to a measured Light Curve (ETR versus
PAR data points) (see also 4.3.2, Light Curve, and 5.5 for theoretical
information). These parameters are:
Fv/Fm x ETR factor/2
Maximum yield of electron- electrons/photons
transport calculated from Fv/Fm
alpha Initial slope of light curve, electrons/photons
related to maximum yield of
photosynthesis
ETRmax Maximum electron transport rate µmole electrons/(m
2
·s)
Ik PAR value of the point of inter- µmole photons/(m
2
·s)
section between a horizontal line
ETRmax and the extrapolated
initial slope.
Light Calibration
Here, a factor can be entered to calibrate a quantum sensor connected
to the PAM-2500 (Factor range: 0.2 to 5). For normal operation of
the quantum sensor of the Leaf Clip Holder 2030-B, the default factor of 1.000 applies.
40
PAMWIN-3 ADVANCED LEVEL CHAPTER 4
Light Offset
The PAR sensor of the 2030-B leaf clip holder works at two ranges
of signal amplification. The Auto Zero function establishes auto-
matically the correct offset values for both ranges: the determination
of zero offset requires that the PAR sensor is kept in the dark. Also,
offset values can be entered manually.
Temperature Offset
<Temperature Offset> can be used to adjust an offset value of an external temperature sensor (offset range is -30 to +30).
ETR Factor
The ETR-Factor corresponds to the fraction of incident photons absorbed by photosynthetic pigments. The factor is required to derive
estimates of electron transport rates from the effective quantum yield
of PS II yield, Y(II) (see Chapter 5.5). The default value for the ETR
Factor is 0.84 which reasonably matches the average fraction of absorbed light in the visible range (400-700 nm) of many green leaves.
AL Current/PAR Lists
All light sources integrated in the PAM-2500 are LEDs which show a
very reproducible relationship between LED current and light output.
Therefore, illumination conditions with PAM-2500 light sources are
well-defined for a particular optical geometry between sample and
fiberoptics exit. For frequently used optical geometries, a set of Current/PAR lists is provided. This greatly facilitates assessment of PAR
and derived ETR values, particularly when online measurement via a
micro quantum sensor is problematic.
Each PAR list consists of three columns: <AL> is the setting number
for the Actinic Light intensity, <Current> represents relative values
for the LED current with the value of 255 corresponding to the maximum current, and <PAR> lists values of PAR in µmol pho-
41
CHAPTER 4 PAMWIN-3 ADVANCED LEVEL
tons/(m2·s). Current values and red Actinic Light intensity are line-
arly related; current values and blue Actinic Light intensity are quasilinearly related.
Default PAR lists show an approximately exponential relationship
between PAR and setting number (Fig. 4.4). Each default PAR list
comes with information on the optical geometry used which can be
viewed by clicking on the comment icon: . To modify the current
or PAR data, click once on the field to be edited, and enter the new
value. New PAR lists can be commented (icon: ), saved (icon:
), and recalled (icon: ).
Note that in all PAR default lists, the PAR value at AL setting <0>
(Actinic Light off) represents the integrated Measuring Light intensity at intensity setting 10 and 100 000 Hz measuring pulse frequency. An “internal PAR value” corresponds to the sum of the Actinic Light intensity plus the integrated intensity of Measuring Light.
The latter is derived from the base value at AL level <0> by taking
Figure 4.4: Red Actinic Light Intensities at Sample Level of a Leaf-Clip
Holder 2030-B.
42
PAMWIN-3 ADVANCED LEVEL CHAPTER 4
into account the frequency and intensity setting of the Measuring
Light.
During setup of PamWin_3, the <default_60.par> file is automatically placed in the <C:\PamWin-3\Data_2500> directory together
with additional PAR lists as specified in Table 4.1. The <default_60.par> data are automatically loaded during the first start of
the PamWin-3 software. The <default_60.par> list corresponds to the
situation at sample level of a 2030-B Leaf-Clip Holder with the fiber
optics fully inserted in the 60° port.
When the file <default_60.par> is not present in the <C:\PamWin3\Data_2500> directory, factory values are used. Thereafter, at program start the most recently used PAR list is loaded.
Mode
In the <Mode> menu, the <VIEW> command switches to off-line
operation: clicking on <MEASURE> reverses the latter action. The
<VIEW> and <MEASURE> commands are equivalent to the
VIEW-mode key in the <General Settings> Window (Fig. 4.3) and
the MEASURE-mode key which is available in the off-line mode.
Table 4.1: Default Red and Blue PAR Lists:
Intensities at Sample Level
File Name Sample Holder Optical Geometry*
default_60.par
Leaf-Clip Holder 2030-B
Distance Clip 2010-A
Arabidopsis Leaf Clip 2060-B
60°
60°, 2 mm distance ring
60°, 6 mm distance ring
default_90.par Leaf-Clip Holder 2030-B 90°, 4 mm distance ring
default_90_2060.par Arabidopsis Leaf Clip 2060-B 90°, 2 mm distance ring
default_DLC.par Dark-Leaf-Clip DLC-8 90°
default_KS.par
KS-2500 Suspension
Cuvette
90° and 4 mm
*Detailed descriptions: see <Comment Files> attached to each PAR list.
43
CHAPTER 4 PAMWIN-3 ADVANCED LEVEL
Service
The <Service> menu is required for firmware update (the software
residing on instrument processors is denoted firmware). New firmware is provided by the Walz Company. To determine your firmware
version, click on the menu item <Read Firmware Version>.
Note: to avoid interruption of communication during firmware update, a stable power supply is mandatory: power failure can lead to
malfunction of the processors, which then need to be re-programmed
at the Walz Company.
To start firmware update, click <Controller Service>. The PAM-2500
contains 2 consecutively programmable processors: RISC and TINY
(a tiny RISC processor).
a) RISC
Sequentially click on Program RISC ¼ Read HexFile ¼
Download HexFile .Then, the actual process of programming is
initiated by clicking on the Program RISC key (this process re-
quires some time). Finally, terminate the procedure by a mouse click
on OK in the <Program RISC> window, confirm with OK the in-
formation on program restart, and once again click OK in the
<PAM-2500 Controller Service> window.
Before proceeding, close and restart the PamWin-3 program!
b) TINY
Programming the TINY processor is similar to the procedure explained for the RISC processor. Start by clicking on
Program TINY . Subsequently, click Read HexFile ¼
Download HexFile ¼ Program RISC . Finalize TINY pro-
gramming by an OK in the <Program RISC> window, the (program
44
PAMWIN-3 ADVANCED LEVEL CHAPTER 4
restart) information window, and the <PAM-2500 Controller Service> window. Close and restart the PamWin-3 program.
<Trigger out with SP> This function puts out a 5 Volts trigger pulse
with each Saturation Pulse. See Section 4.3.1.7 for a description of
the trigger pins at the AUX connector and configuration of the trigger
pulse.
Help
In the Help menu, checking Tooltips activates the PamWin-3
tooltips function described earlier (Section 4.1). Clicking on Info
displays information on the current PamWin-3 version.
4.3.1.2 Measuring Light Frequency
Measuring Light frequencies are adjusted in the <General Settings>
window (see Fig. 4.3): icons and settings are summarized below.
Table 4.2: Measuring Light Frequency
Icon Range
MF-L.
Low Measuring Light frequency 10 - 5 000 Hz
MF-H.
High Measuring Light frequency 1 000 - 100 000 Hz*
* The instrument’s maximum frequency of 200 000 Hz is effective in
the <Fast Acquisition Analysis Mode> in triggered Fast Kinetics only.
4.3.1.3 Induction Curve Parameters
PamWin-3 defines the time course of fluorescence Induction Curves
by three Induction Curve parameters (Fig. 4.3):
Delay, seconds
: determines the dark interval between Fv/Fm deter-
mination and onset of actinic illumination. Default interval is 40 s.
45
CHAPTER 4 PAMWIN-3 ADVANCED LEVEL
Clock, seconds: defines the interval between Saturation Pulse analy-
ses during actinic illumination.
Width, seconds
: specifies the length of time of Actinic illumination.
All three time intervals are adjusted using upward and downward arrow keys, or by double click on the interval number followed by
manually entering the new time value. The AL-Width is automatically adjusted to equal a multiple of clock intervals plus 10 seconds.
The extra 10 seconds are assigned to the initial and last Saturation
Pulse applied during actinic illumination. Example: for a clock interval of 50 s, possible illumination times (<Width>) are 60 s, 110 s,
160 s …
4.3.1.4 Global Settings
The PamWin-3 software allows to adjust numerous instrumental settings, so that an almost infinite number of setting combinations results. Therefore, PamWin-3 offers the possibility to load a default set
of standard settings and to save any particular set of settings which
can be recalled later to carry out the same experiment with identical
settings.
Four buttons manage the PAM-2500 <Settings> (see Global Settings
in Fig. 4.3).
Default Installs default settings (File:
<Walz2500.DEF>).
Open User Settings Loads previously stored settings except
the Zoff which is set to zero when new
<Settings> are loaded.
Save User Settings Saves the current setting to a file with for-
mat <filename.DEF> except the Zoff.
46
PAMWIN-3 ADVANCED LEVEL CHAPTER 4
Write To Report Writes the settings to the current Report
(see 4.4.3.1 for abbreviations used by a
Report).
Other Commands
Zoff The <Zoff> (Zero Offset) serves to suppress a “background
signal” which is not originating from the investigated sample. It is
subtracted from all fluorescence signals. Background signals can
arise from traces of scattered Measuring Light, which reaches the
photo-detector. While this normally does not play a role in experiments with leaves, it may matter with suspensions at low chlorophyll
content. In this case, the Zoff is determined with the cuvette containing pure suspension medium by pressing Zoff . The <Zoff> can also
be entered manually.
Exit PamWin shuts down PamWin-3
4.3.1.5 PAR Sensor Control
Checking the <PAR Sensor Control> results in reading of data from
an external PAR sensor. When the <PAR Sensor Control> is unchecked, PAR values are derived from the active internal light list.
Internal Actinic Light arriving laterally at the entrance optics of the
micro quantum sensor of the Leaf-Clip Holder 2030-B is not properly
measured (Section 3.4.2.3). Therefore, when this sensor is connected,
it should be disabled via <PAR Sensor Control> for applications involving actinic illumination by internal light sources (e.g. recording
of Light Curves).
4.3.1.6 Fo, Fo’
Clicking the Fo icon (see Fig. 4.3) takes the present Ft fluorescence
level as Fo level fluorescence that corresponds to the minimum fluo-
47
CHAPTER 4 PAMWIN-3 ADVANCED LEVEL
rescence of a dark-acclimated sample. Hence, an Fo measurement occurs without subsequent Fm determination, i.e., without exposing the
sample to a saturating flash.
The C/Fo value indicates the relative contribution of a constant fluorescence fraction (which likely originates in PS I) to the measured Fo
level fluorescence. The assessment of C/Fo requires that the Fo’
mode is activated and that non-photochemical fluorescence quenching decreases the Fo’ below the Fo level (see Section 5.3 for details).
Fo’ The Fo’ is the measured
Fo’ level fluorescence. Checking the
Fo’ box (Fig. 4.3) activates the Fo’ measuring mode. This means that
a 5 seconds interval succeeds each Saturation Pulse during which the
Actinic Light is switched off and <PS I light> is turned on. The PS I
light quickly empties the PS II electron acceptor pool and, hence,
opens the PS II reaction centers so that photochemical fluorescence
quenching becomes maximal. The intensity of PS I light can be adjusted in the <General Settings> window but the post-pulse time interval for far-red illumination is fixed.
˜Fo represents the calculated
Fo’ level fluorescence. The ˜Fo’ is assessed from Fo, Fm, and Fm’ data according to Oxborough and
Baker (1997; for details see Section 5.1.2).
4.3.1.7 Tab Bar
Each active window of PamWin-3 appears as a tab in the Tab Bar,
and clicking on a tab moves the respective window to the foreground.
4.3.1.8 Pulses, Short Term Illumination
The advanced level offers additional ways of sample illuminations
but also the possibility to apply a trigger signal to control an external
device (Fig. 4.3).
48
PAMWIN-3 ADVANCED LEVEL CHAPTER 4
~ 3
~ 4
~ 5
~ 6
~ 1
~ 2
~ 7
~ 8
1. DGND (digital ground)
2. UBatt (battery voltage)
3. GND (ground)
4. DA out (analog output)
5. DGND (digital ground)
6. Trigger Out
7. +5 V
8. -5 V
~ 3
~ 4
~ 5
~ 6
~ 1
~ 2
~ 7
~ 8
~ 3
~ 4
~ 5
~ 6
~ 1
~ 2
~ 7
~ 8
1. DGND (digital ground)
2. UBatt (battery voltage)
3. GND (ground)
4. DA out (analog output)
5. DGND (digital ground)
6. Trigger Out
7. +5 V
8. -5 V
Figure 4.5: AUX Socket.
Short-term events ranging from a few µseconds to 1000 ms are:
ST Single turn-over flash with flash lengths between 5 µs and
50 µs, and a PAR value of about 100 000 µmol/(m2·s).
MT Multiple turn-over flash with flash lengths between 1 ms and
300 ms, and PAR value up to 25 000 µmol/(m2·s).
TR Pulse 5 Volts trigger pulses delivered to the AUX socket.
Pulse times range from 10 µs to 1000 ms (Fig. 4.5).
To access time settings of short-term illumination, open the <Pulse
Widths> window by a click with the right mouse key on ST , MT ,
or TR Pulse .
When in the <Pulse Widths> window S+H off is checked, the sample-and-hold amplifier of the PAM-2500 is switched off for the duration of the ST flash. The deactivation of the sample-and-hold amplifier prevents artifactual signals arising from the extremely high light
intensity of the flash. The S+Hold off time can be extended beyond
the ST flash time by selection of an additional time from the drop
down list Extended S+H off time.
TR The key permits manual control of the trigger out signal.
The following two buttons are used for illumination in the seconds
range in combinations with Saturation Pulse analysis.
49
CHAPTER 4 PAMWIN-3 ADVANCED LEVEL
AL + Y The command illuminates the sample with Actinic Light as
defined in <General Settings> and thereafter carries out a Saturation
Pulse analysis. This command is available for Actinic Light widths of
3 s or longer. With appropriate choice of AL Intensity and Width,
AL+Y measurements can provide essential information on the photosynthetic performance of a sample and is well suited for the screening
of deficiencies.
FR + Y This command works similarly as the <AL + Y> command
except for the Actinic Light being replaced by PS I light.
4.3.1.9 Mode
The mode area in the <General Settings> window (Fig. 4.3) represent
a hub leading to different program levels, that is, the three PamWin-3
measuring modes (Field Screen, Advanced SP-Analysis and Advanced Fast Acquisition) and the PamWin-3 <View> mode for offline data evaluation. In the Fast Acquisition mode, emphasis is on the
recording of kinetic changes. While the same multiple turnover saturating light pulses (MT) can be applied as in the SP-Analysis mode,
there is no automatic determination of fluorescence parameters. An
exception is the <Fo, Fm> determination, with calculation of Fv/Fm.
4.3.1.10 Clock Event
In total, 6 different events can be repetitively triggered by the clock
function. All events are defined under <General Settings> except for
the light curve which is defined on the <Light Curve> window.
Theoretically, the interval time between events ranges from 3 to 900
seconds. Practically, the minimum interval needs to be longer than
the event time plus some extra time to allow for data processing. The
clock-triggered events are:
50
PAMWIN-3 ADVANCED LEVEL CHAPTER 4
SAT-Pulse: Saturation Pulse with quenching analysis.
AL: illumination with Actinic Light for a time period de-
fined under General Settings/Act.Light/Width.
AL + Y: illumination with Actinic Light for a time period de-
fined under General Settings/Act.Light/Width, followed by a Saturation Pulse with quenching analysis.
FR + Y: illumination with PS I light for a time period defined
under General Settings/PS I Light/Width, followed
by a Saturation Pulse with quenching analysis.
Light Curve: Light Curve defined in the Light Curve Edit window.
Slow Induc. Fluorescence Induction curve (Kautsky-effect), as
defined under General Settings/Slow Induction.
4.3.1.11 New Record
In the SP-Analysis Mode, data are grouped as <Records>. A new
<Record> is started by clicking on the New Record button which
becomes activated at the first <Fo, Fm> determination after program
start. Starting a new <Record> does not interrupt continuous recording in the <Slow Kinetics> graph.
Usually, a <Record> starts with an <Fo, Fm> determination using a
dark-adapted sample. The Fo and Fm level fluorescence enters the
calculations of many fluorescence ratio parameters (see Chapter 5). It
is therefore important to carry out proper <Fo, Fm> determinations
and to correctly organize the recording of Saturation Pulse data (i.e.
assigning one <Record> to each sample).
51
CHAPTER 4 PAMWIN-3 ADVANCED LEVEL
4.4 SP-Analysis Mode
4.4.1 Slow Kinetics Window
The primary function of the <Slow Kinetics> window is real-time
display of fluorescence kinetics, Ft, and of fluorescence ratio parameters derived from Saturation Pulse analysis. The new graphical elements of the <Slow Kinetics> window are displayed in Figure 4.6.
The figure does not show the elements already introduced in the previous section (<General Settings>).
The <Menu Bar> of the <Slow Kinetics> and <General Settings>
windows are similar except the <File> menu which contains as addi-
Figure 4.6: Slow Kinetics Window.
52
PAMWIN-3 ADVANCED LEVEL CHAPTER 4
tional commands <Print Graph> and <Save as pws-file>. Execution
of the latter command saves the current Ft fluorescence trace in the
“P
amWin Slow Kinetic” format (*.pws). In the <View> mode, the Ft
file can be opened and converted into text format to be imported by
other programs.
4.4.1.1 Graph Icons
The three graph icons outlined in Fig. 4.6 control display of the graph
grid (left icon), automatic scaling of both graph axes (central icon),
and printing of the current graph (right icon).
4.4.1.2 Graph Y, X
The left and right numerical display fields (Fig. 4.6) indicate the x
(time in s) and y (relative units) coordinates, respectively, of the cursor position in the graphics field.
4.4.1.3 Save Ft
The disk icon ( , see Fig. 4.6) is identical to the <Save as pws-
file> command in the <File> menu. The document icon ( ) opens
the comment file associated with the most recently saved Ft fluorescence trace.
4.4.1.4 Display Control
Display of the various fluorescence parameters are controlled by
checkboxes (see <Display Control>, Fig. 4.6). The data points and
background colors of the corresponding checkbox labels have identical colors.
53
CHAPTER 4 PAMWIN-3 ADVANCED LEVEL
4.4.1.5 X-Axis Scaling/Zoom In
Clicking on the arrow directing to the left (◄, see Fig. 4.6) increases
the total x axis time range by a factor 2, while the total x axis time
range is divided by 2 when the arrow directing to the right (►) is
clicked.
In addition to the x-axis scaling buttons, both, x- and y-scaling can be
modified to magnify a particular part of the graph: to “Zoom in”,
click with the left mouse button on the left upper border of the target
area, move the mouse with the left button pressed down to the right
lower border of target area, and release the mouse button (see Fig.
4.6). A right mouse click anywhere on the graph field restores the
original display.
The currently displayed graph can be moved vertically by pressing
<Shift> key and gripping the graph with the left mouse button. With
the <Ctrl> button depressed, the mouse moves the graph horizontally.
4.4.1.6 Slow Kinetics Control
The drop-down menu in the <Slow Kinetics Control> area (Fig. 4.6)
allows to choose between a manually controlled recordings
(Manual), automated recordings of dark-light fluorescence induction
curves (Ind.Curv.) or dark-light induction curves followed by an
extended dark recovery time, during which the relaxation of nonphotochemical quenching is monitored (Ind.+Rec.).
The Start and Stop buttons in the < Slow Kinetics Control> area
start and end a <Record>. Several <Records> can be stored in one
<Report>.
Recordings of Ind.Curv. And Int.+Rec. are always initiated by
automated <Fo, Fm> measurements and the Fm serves for scaling the
Ft signal between 0 and 1 (ordinate scale corresponding to Ft/Fm).
54
PAMWIN-3 ADVANCED LEVEL CHAPTER 4
The same scale also applies to the fluorescence ratio parameters, except for NPQ, which can assume values above 1. Therefore, NPQ/4
values are plotted.
Manual recordings can be carried out without <Fo, Fm> measurements. In this case, the Ft signal is recorded using the original voltage
scale. For full quenching analysis, however, an <Fo, Fm> determination is required (cf. Chapter 5).
4.4.2 Light Curve
The <Light Curve> window allows exposure of samples to a series of
illuminations which may differ in duration as well as in intensity. At
the end of each illumination step, a Saturation Pulse analysis is carried out. The <Light Curve> chart plots Saturation Pulsed analysis
data against PAR. Often, such illumination programs consist of light
steps with identical time intervals and increasing intensities. The
graphical representation of the results is denoted Rapid Light Curves.
For more information on Rapid light curves see Section 5.5.
4.4.2.1 Display Control
Light Curves are plotted with PAR (in µE/(m2·s)) as the abscissa parameter. Ordinate data are selected by checking ETR or Fluoresc.
in the Display Control field (see Fig. 4.7): when Fluoresc. is selected, fluorescence ratio parameter are graphed. With ETR checked,
relative electron transport rates derived from Y(II) and the flux of
photons absorbed by PS II is drawn (equation: Section 5.4). A plot of
photosynthetic fluxes versus light intensity is called light response
curve. Compared to traditional light curves, which require steady
state conditions for each light step, the ETR versus PAR plots often
employ rather short intervals per light step and, therefore, are also
called “Rapid Light Curves” (see 5.5.1 for papers on light curves).
55
CHAPTER 4 PAMWIN-3 ADVANCED LEVEL
Figure 4.7: Light Curve Window.
4.4.2.2 LC (Light Curve) Control
The Start and Stop buttons initiate and manually terminate a
Light Curve recording. Normally a Light Curve recording is terminated automatically following application of the last pre-programmed
Saturation Pulse.
56
PAMWIN-3 ADVANCED LEVEL CHAPTER 4
4.4.2.3 Light Curve Edit
Programming of the Light Curve parameters are done in the <Light
Curve Edit> window (see Fig. 4.7). Up to 20 illumination steps can
be programmed. A particular Light Curve illumination program can
be saved as Light Curve Program and loaded again at any later time
using the icons on the top of the <Light Curve Edit> window. The
default light curve file is denoted <default.lcp>.
Programmable parameters are the PAM-2500 Actinic Light intensity
setting (1 to 20 in Intens. column) and the time intervals of a light
intensity step (in 10 seconds intervals, Time/10s). To change settings, click in the target field once and type in the new number. The
change becomes effective upon the next click. To set the same illumination interval for all steps, check <Uniform time>, before entering the desired time and terminating with another click. A zero in the
Time/10s column ends exposure (e.g., when “0 seconds” is attributed to step 6, only steps 1 to 5 will be executed.) When the 0 is entered, <Uniform time> should be disabled.
The light intensities used as x-values correspond to those of the selected internal PAR list. It is not recommended to measure the PAR
online (see Section 3.4.2.3). If the Leaf-Clip 2030-B is connected, the
PAR sensor should be disabled under General settings.
4.4.2.4 Light Curve Fit
The equation derived by Eilers and Peeters (Ecological Modelling 42
(1988) 199-215) is used to describe rapid light curves. From the results of curve fitting, three cardinal parameters are calculated (see
Fig. 4.7):
alpha: The initial slope of the rapid light curve which is related to
the maximal quantum yield of PS II electron transport under light
limited conditions (unit: electron/photon).
57
CHAPTER 4 PAMWIN-3 ADVANCED LEVEL
ETRmax: The maximal relative electron transport rate reached during Light Curve recording (unit: µmol e
-
/(m2·s)), which reflects the
light saturated capacity of the sample.
Ik: The light intensity at which the alpha and ETRmax lines intersect
(unit: µmol photons/(m
2
·s)), which may be considered a proxy for the
PAR where saturation sets in.
In the Light Curve Fit window, besides the three cardinal parameters
obtained from the fitting procedure, also the Fv/Fm x ETR factor/2
is displayed. This is meant to remind that Fv/Fm corresponds to the
maximal quantum yield of charge separation at PS II reaction centers,
from which the parameter alpha can be predicted, when the ETR factor (normally 0.84) and the fraction of quanta distributed to PS II
(normally 0.5) are considered. The Fv/Fm x ETR factor/2 tends to
be somewhat larger than alpha, due to some decrease of Y(II) at low
light intensities.
4.4.3 Report
Generally, a Report file consists of a list of data measured by Saturation Pulse analysis. The data are organized by date/time and columns
of the various measured parameters. The user may add comments
with additional information on the particular measurements. A Report
file can contain several Records, each of which normally represent a
series of Saturation Pulse analyses with the same sample.
4.4.3.1 Report Data Management
A Report file can contain up to 1600 Saturation Pulse analyses. The
percentage of file space used is indicated numerically and graphically
(see <Report Data Management> in Fig. 4.8). The window area <Report Data Management> also provides icons to access the current
58
PAMWIN-3 ADVANCED LEVEL CHAPTER 4
comment text ( ), to open a previously saved Report (), and to
save the current Report ( ).
The <Save Report> command saves the current Report data in PamWin-3 format using the extension <rpt> (e.g., filename.rpt). File
name and location can be selected. Default directory is
C:\PamWin_3\PamWin-3\Data_2500. The <Open Report> command
activates the <View Mode> in which previously saved files can be
opened and processed. Switching from the <Measure Mode> to the
<View Mode> automatically saves the current Report data in the file
<PamWin.RPT>. The data of <PamWin.RPT> are loaded when returning to the <Measure Mode>, or after program start.
The <Save Report> and <Open Report> commands are also available
in the <File> menu which also provides the <Clear Report> commands to delete the current Report data. The <Clear Report> also initiates a new Report by writing a line of PAM-2500 <Settings> on top
Figure 4.8: Report Window.
59
CHAPTER 4 PAMWIN-3 ADVANCED LEVEL
of the new line: the line of PAM-2500 <Settings> includes 16 parameters (see Table 4.3). Similarly, the <Write to Report> command
on the <General Settings> window creates PAM-2500 <Settings>
line.
Table 4.3: Report of Instrument Settings
Abbreviation Parameter
AI Actinic Light Intensity
AW Actinic Light Width
BI Blue Light Intensity
BW Blue Light Width
D Damping
FI Far-red Light Intensity
FW Far-red Light Width
G Gain
H High Measuring Frequency
ID Slow Induction Delay
II Slow Induction Clock
IS Slow Induction Actinic Light Width
L Low Measuring Frequency
MI Measuring Light Intensity
SI Saturating Pulse Intensity
SW
Saturating Pulse Width
4.4.3.2 Record Header and Last Line
First Line: Record Title
The first line of a Record contains the Record title. A Record title is
written automatically, if entered in the text field above the graphics
chart in the <Slow Kinetics> or <Light Curve> window. It can also
be written manually. In this case, just like with any other text comment line, the text must begin with a semicolon (;) for distinction
from data lines.
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PAMWIN-3 ADVANCED LEVEL CHAPTER 4
Second and Third Line: Fo, Fm
The second and third Record lines contain data related to an
<Fo, Fm> determination, which normally should be carried out at
start of a new Record. The abbreviations <IC> and <IC+> in the
second line indicate that the measuring programs <Induction Curve>
and <Induction Curve and Recovery>, respectively, have been
started.
Notes: (1) when <Fo, Fm> determination is not the first Saturation
Pulse analysis of a Record, and when the graphics text field is left
blank, lines 1 to 3 are reduced to the date of the experiment, (2) an
<Fo, Fm> determination does not start a new Record, (3) a <Fo, Fm>
determination during a Record is not indicated in the Record, but the
Fo and Fm values are used for calculations of fluorescence ratio parameters.
Last Line: Saving of Slow Kinetics file
If the Slow Kinetics measured during the Record was saved, the corresponding file name is documented in the last line.
4.4.3.3 Record Columns
In the measuring mode, a Record chart contains 12 columns. More
data are available in the <View> mode. The subsequent comments on
Report columns use the column numbering depicted in Fig. 4.8.
1 Date and time.
2 Sequential numbering of Saturation Pulses. The number <1> is
omitted if the first Saturation Pulse was applied to determine
<Fo, Fm>.
3 Setting of Measuring Light intensity
4 Temperature (°C)
5 Actinic light intensity (PAR, µmol photons/(m
2
·s)).Light inten-
sity (PAR, µmol photons/(m
2
·s).
61
CHAPTER 4 PAMWIN-3 ADVANCED LEVEL
6 Fo (header lines) and F (subsequent data lines) in Volts.
7 Electron transport rate (ETR, µmol electrons/(m
2
·s))
8 Maximum (header lines) and effective photochemical quantum
yield (subsequent data lines) of PS II
9 Coefficient of photochemical fluorescence quenching (qP). Set
to 1.000 in the absence of appropriate Fo and Fm data.
10 Coefficient of non-photochemical fluorescence quenching. Set
to 0.000 in the absence of appropriate Fo and Fm data.
11 Fm (header lines) and Fm’ (subsequent data lines) in Volts.
12 Fo’ data as determined with the help of far-red illumination. If
the Fo’-mode is not selected, column 12 stays blank.
13 Battery voltage (Volts).
4.5 Fast Acquisition Mode
The <Fast Acquisition Mode> of PamWin-3 allows to study fluorescence kinetics in the sub-msec to sec range using a maximum time
resolution of 10 µs. This mode is specialized for well-defined triggered illumination by red, blue or far-red light. The measured fluorescence changes can be averaged and in this way an excellent signalto-noise ratio can be obtained at high time resolution even with
weakly fluorescent samples. Except for calculation of Fv/Fm, no SP
analysis is carried out. According to the different mode of analysis,
three windows present in the SP-analysis mode (<Slow Kinetics>,
<Light Curve>, and <Report>) are replaced by the <Fast Kinetics>
and <Fast Settings> windows. The new features of these windows
will be introduced next.
4.5.1 Fast Settings
The <Fast Settings> window represents the user surface to program
timing and light conditions of fast kinetics recordings. In this window, the various tools required to design a particular fast kinetics
62
PAMWIN-3 ADVANCED LEVEL CHAPTER 4
trigger program are grouped in different window areas according to
their functionality (Fig. 4.9).
4.5.1.1 Basic Settings
Rate/Points The sampling rate corresponds to the time interval between two consecutive ML pulses. The rate thus determines time
resolution. The total time interval of a <Fast Kinetics> recording is
determined by the product of the sampling rate and the number of
points. A trigger signal cannot be programmed at times exceeding
Figure 4.9: Fast Settings Window.
63
CHAPTER 4 PAMWIN-3 ADVANCED LEVEL
32 000 points, while the overall <Fast Kinetics> may comprise up to
128 000 points. For example, a <Fast Kinetics> can be composed of
an initial fluorescence rise, induced by triggered illumination, which
is followed by a fluorescence decay curve.
Target Averages The “Target Averages” correspond to the number of Fast Kinetics recordings that will be averaged, when Averaging is enabled via the <Averaging> checkbox in the <Fast Kinetics>
window. Consecutive recordings can be either triggered manually
(Fast Kin. button / Start button) or via the Clock.
4.5.1.2 Menu Bar
The composition the <Menu Bar> in the <Fast Kinetics> and the
<SP-Analysis> mode is mostly identical (Section 4.3.1.1) except the
items listed in the <Window> menu which correspond to the windows available in the current mode.
4.5.1.3 Trigger Settings ( I ) and ( II )
To configure trigger signals, 6 different tool icons are provided (see
Figs. 4.9 and 4.10). The 3 left-most icons in Fig. 4.10 represent
commands which affect the entire trigger signal but the three icons on
the right hand side configure signal changes of various lengths which
is shorter than the total trigger interval.
Figure 4.10: Trigger Tool Icons.
64
PAMWIN-3 ADVANCED LEVEL CHAPTER 4
Eight different events can be triggered (see Trigger Settings ( II ) and
Table 4.4). Among these events, the ST (single turn-over flash) needs
to be very short to turn over the PS II reaction center only once and,
hence, only the <High Pulse> and <All Low> buttons are available
for configuring the ST.
All other events can be triggered by pulses and by signal low/high
shifts. To set a pulse:
Table 4.4: Trigger Chart: Checkbox Panel
Checkbox
Label
Action Comments
AL(Red) Red Actinic Light
Intensity - as defined for <Act.
Light> in <General Settings>
FR/BL
Far-red light/Blue
light
FR/Blue selection: <General Settings>. Intensity as defined for
<PS I Light> in <General Settings
>
F ML Measuring Light
Frequencies (MF-L. and MF-H.)
and intensity: <General Settings>.
Measuring Light settings at kinetic
s
start are controlled by ML and
MF-H buttons.
MT
Multiple turn-over
flash
Intensity - as defined for <Sat.
Pulse/MT> in <General Settings>
TR Trigger out See Section 4.3.1.7
MF-max
Maximum frequency of Measuring Light
Frequency as defined in <Fast Settings> window (MF-max)
S&H off
Sample and hold
circuit off
Suppresses artifacts caused by extremely strong and fast intensity
changes of non-modulated light
ST
Single turn-over
flash
Always at maximum intensity
(about 125 000 µE/(m
2
·s)
65
CHAPTER 4 PAMWIN-3 ADVANCED LEVEL
a) Select an event by clicking on its checkbox label (Fig. 4.9.
Trigger Setting ( II )). The event is selected when its label is
typed white on blue background.
b) Activate event by clicking the box associated with the label.
c) Click on <High Pulse> icon.
d) Select time interval in drop-down menu. Note that the <ST
Width> menu applies for ST only, but pulse lengths of the
other events must be selected in the <Pulse Width> menu.
Also, the minimum pulse width depends on the sampling rate
selected (Fig. 4.9. Basic Settings).
e) Click in the field below <Trig on, µs> (see Fig. 4.9. Trigger
Setting ( II )) and type in the time point of pulse start in µs.
Hitting the <Enter> key results in the digital display of the
pulse end time in the neighboring data field (calculated with
the interval selected above), and in the graphical display in
the <Trigger Graph>.
Repeating step e) places an additional pulse on the same trigger line.
For the same event, pulse intervals may vary: simply visit step d) before proceeding to e). To move to the next event, simple select and
activate the event (steps a) and b), respectively).
Longer-term triggering is achieved by using the <Toggle Low/High>
tool (Fig. 4.10). Basically, the procedure is identical to the setting of
trigger pulses but in step c) the <Toggle Low/High> icon must be selected and, in step e), the setting of the <Trig on, µs> time point does
not automatically results in an entry in the <Trig off, µs> field. Consequently, the trigger signal can be
- switched only from low to high (no entry in the <Trig off, µs>
field)
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PAMWIN-3 ADVANCED LEVEL CHAPTER 4
- switched only from high to low (no entry in the <Trig off, µs>
field followed by the <Invert All> command. Fig. 4.10.), or
- switched from low to high and then back at the time points en-
tered in the <Trig on, µs> and <Trig off, µs> fields, respectively.
Table 4.5: Fast Settings - Trigger Program Tools*
Action
Active
Icon
Key Combination Parameters
Main Trigger Chart
High
pulse*,**
<Shift> + <right mouse key> All
Low
pulse*,**
<Shift> + <right mouse key> All
Switch to
high**
<Shift> + <left mouse key> All but ST
Switch to
low**
<Shift> + <right mouse key> All but ST
All low
All
All high
All but ST
Invert
levels
First, select event
to be triggered
(click on checkbox label)
All
Pretrigger Chart
Toggle between always
high and always low
<right mouse key> All but ST
* For all events, <Pulse Width> applies except the ST interval
which is determined by <ST Width>. Time points in µseconds
of trigger on and off events are numerically displayed (see Fig.
4.9, Trigger Settings ( II ) ).
** Trigger setting via these shortcuts does not require selection of
a trigger event in the checkbox panel.
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CHAPTER 4 PAMWIN-3 ADVANCED LEVEL
Additionally, trigger pattern can be created by directly manipulating
the <Trigger Chart> using the mouse pointer as outlined in Table 4.5.
The graphical editing of trigger lines is facilitated by the numerical
displays of the mouse cursor position (numerical displays below
<Trigger Tool Icons>).
For accurate setting of time points, the x axis can be enlarged as outlined in Table 4.6 which also comments on how to shift the x axis after enlargement.
Trigger files can be saved and recalled using the appropriate icons in
the <Trigger Settings ( I )> field: and , respectively. Click-
ing the Default button in <Trigger Settings ( I )> opens the
<Poly300ms.FTM> which is one of the predefined trigger files of
PamWin-3.
When Enable Pretrigger is checked (Trigger Settings ( II ), Fig.
4.9), a display field appears below the checkbox, which is associated
with a drop down list of pretrigger time intervals. Also, a small chart,
Table 4.6: Fast Settings - Chart Manipulation
Action Key Combination
Enlarge x-axis
scale
Click with <left mouse key> at lower x
limit, while keeping the mouse key depressed move cursor to the upper x limit,
release key.
Switch between
enlarge x-axis and
shift x-axis
Single <right mouse key> on trigger chart
Shift x-axis
Click on chart with <left mouse key>, hold
mouse key down and move cursor horizontally
Full x-axis scale <Auto button>
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PAMWIN-3 ADVANCED LEVEL CHAPTER 4
entitled <Pre>, left of the <Trigger Graph> becomes apparent. In the
<Pre> chart, a trigger signal can be toggled between low and high by
left-clicking on trigger line. A pretriggered event is active for the pretrigger time interval selected. The pretrigger interval is immediately
followed by the fast kinetics. Note that, different to the main trigger
pattern, the pretrigger settings are not set <All Low> by the
Clear All button.
Zero Time Shift (Trigger Settings ( II ), Fig. 4.9) right-shifts the
zero point of the time axis so that recording of a fast kinetics starts at
negative time values but triggered action can be initiated at zero or
positive time points. Zero time shift is advantageous when fluorescence data are plotted against a logarithmic time scale. To adjust the
zero time shift, double-click on the number displayed below the
phrase <Zero Time Shift> and enter the new time value.
4.5.1.4 Light & Pulses
In the <fast acquisition> mode, two buttons are available which first
start an illumination with Actinic or far-red light and then initiate the
execution of a fast kinetics (Fig. 4.9):
The AL + F.K. command starts an Actinic illumination and then
executes the current trigger pattern.
The FR + F.K. is similar to the previous command except that far-
red illumination is given instead of Actinic Light.
Note: the illumination regimes of AL + F.K. / FR + F.K. differ
from the pretrigger illumination (see previous Section) because the
Actinic/far-red light stays on during the fast kinetics. The time interval for Actinic/far-red light must be defined in <General Settings>;
the fast kinetics will be performed 2 seconds before the time interval
ends.
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CHAPTER 4 PAMWIN-3 ADVANCED LEVEL
4.5.1.5 Start Fast Kinetics
Fo, Fm Clicking the <Fo, Fm> button starts determinations of Fo
and Fm values, calculation of the Fv/Fm and entry of the data in the
current Report as described in Section 4.2.1.3.
_Start_ The command starts immediate execution of the current
trigger pattern.
Clock Three different events can be triggered by the <Clock> function in the <Fast Kinetics> mode: fast kinetics, fast kinetics in the
presence of illumination with Actinic Light, and fast kinetics in the
presence of illumination with far-red light. The three events are
equivalent to the commands <Start>, <AL + F.K.>, and <FR + F.K.>
(see above). The clock interval can be set using the up and down arrows, but the interval can also be entered manually after double-click
on the displayed time interval.
4.5.2 Fast Kinetics
The data acquired in the <Fast Kinetics> mode are displayed in the
<Fast Kinetics> window. Most options available for axis control in
the <Slow Kinetics> Window (Section 4.4.1) also work with the
<Fast Kinetics> window. The new features of the <Fast Kinetics>
graphics display are:
- Control of y-axis scaling by up ( ▲ )/down ( ▼ ) arrows, and by
Auto button (Fig. 4.11, Axis Control).
- Toggling option to switch between linear and logarithmic x-axis
scaling (see Log checkbox Fig. 4.11).
- Keep Scale function to fix y-axis scaling when viewing a series
of fast kinetics recorded at different y-axis scales (Fig. 4.11,
y-axis scaling).
70
PAMWIN-3 ADVANCED LEVEL CHAPTER 4
More options of the <Fast Kinetics> window are explained subsequently.
4.5.2.1 Cursor Coordinates
Ordinate and linear abscissa data of the current cursor position are
numerically displayed for evaluations of the fluorescence trace.
4.5.2.2 File Names
Each <Fast Kinetics> is saved using the file name format:
F_data_time_0. The complete list of <Fast Kinetics> is available by
a left-click on the single downward-directing arrow: click on the file
name to view the kinetics traced. To view kinetics sequentially, select
Figure 4.11: Fast Kinetics Window.
71
CHAPTER 4 PAMWIN-3 ADVANCED LEVEL
a file name in the list of <Fast Kinetics>, use the Up/Down arrow pair
to the right of the file name display, or the up/down arrows on your
keyboard, or the mouse wheel.
4.5.2.3 File Menu
<Fast Kinetics> which are not saved will be lost at closure of Pam-
Win-3. Kinetics can be saved as Pam Fast Kinetics files (*.pfk) using
the floppy disk icon in the <File Menu> (see Fig. 4.11). Also, data
can be exported (icon below floppy disk icon) in ASCII or Unicode
formats which are importable by most spreadsheet and graphing programs.
4.5.2.4 Fast Kinetics
A number of elements to manage fast kinetics are pooled in the <Fast
Kinetics> panel (see Fig. 4.11).
Copy Trigger from Fast Kin. Clicking this icon overwrites the
current trigger pattern with the trigger pattern associated with the currently displayed kinetics. The feature aids to reproduce kinetics.
Fast Settings The command switches to the <Fast Settings> win-
dow.
Average Points The figure displays corresponds to the number of
points averaged for graphical display. The “graphical averaging”
does not affect the originals data.
Start The command starts immediate execution of the current trig-
ger pattern.
Auto ML On If checked, the Measuring Light is switched at start of
a <Fast Kinetics>.
72
PAMWIN-3 ADVANCED LEVEL CHAPTER 4
Averaging When <Averaging> is active, the mean of consecutively recorded <Fast Kinetics> will be calculated. The number specified for <Target Average> in the <Fast Settings> window determines
the maximum number of <Fast Kinetics> spectra to be averaged.
With <Averaging> is switched on, the <Clock> triggers only the
number of <Fast Kinetics> specified as <Target Average> so that the
entire averaging process can carried our automatically.
Table 4.7: Default Trigger Pattern < Poly300ms.FTM>
SETTINGS COMMENT
START CONDITION
Auto ML
ML
OFF
OFF
switch on Measuring
Light by <F ML> trigger
command
MF-H
ON
always use high frequency Measuring Light
MF-max
200 kHz
use highest frequency for
Measuring Light for best
time resolution
Zero Time
Shift
10 000 µs
start Record 10 ms before
fluorescence rise
TRIGGER PATTERN
F ML
(trig. high)
-1000 to 300 000 µs
start Measuring Light 1
ms before <MT> to establish Fo, switch off at end
of <MT>
MT
(trig. high)
0 to 300 000 µs
expose sample to a 300
ms <MT>
MF-max
(trig. high)
0 to 300 000 µs
use maximum Measuring
Light frequency while
<MT> on
S&H off
(trig. high)
-10 to 10 µs
299 990 to 300 010 µs
switch off sample & hold
amplifier at start and end
of <MT>
73
CHAPTER 4 PAMWIN-3 ADVANCED LEVEL
4.5.2.5 Polyphasic Fluorescence Rise
Here, the default trigger pattern of PamWin-3 (<Poly300ms.FTM>)
is used to demonstrate aspects of trigger design for <Fast Kinetics>.
The <Poly300ms.FTM> trigger pattern is shown in Fig. 4.9, and a
fluorescence curve elicited by this trigger pattern is shown in
Fig. 4.11.
Details of the <Poly300ms.FTM> trigger pattern are summarized in
Table 1. In short, the triggering results in a <Fast Kinetics> which
establishes a baseline in the absence of Measuring Light at negative
time values. Shortly before time “zero”, high frequency Measuring
Light is switched on the establish the Fo level fluorescence. At time
zero, a polyphasic fluorescence kinetics is initiated by a strong multi
turn-over flash and, simultaneously, the time resolution of measurement is further increased by switching to maximum frequency of
Measuring Light. Figure 4.11 also depicts Fo and Fm level fluorescence which were established by an <Fo, Fm> determination prior to
recording a <Fast Kinetics>.
Generally, to become experienced with PamWin-3 trigger management, it is recommended to start with one of the trigger files provided
by the software and then modify the default settings.
4.6 View Mode
The <View Mode> of PamWin-3 is dedicated to analyses of fluorescence measurements. Three windows are available in the <View
Mode>: the <Report>, <Slow Kinetics> and <Light Curve> windows. The operation of the latter two windows is as described previously (Sections 4.4.1 and 4.4.2).
The <Report> window in the <View Mode> provides new tools for
data management and navigation; the <Icon Bar>, the <Sidebar>, and
a panel for data selection (Fig. 4.12).
74
PAMWIN-3 ADVANCED LEVEL CHAPTER 4
Figure 4.12: View Mode Window.
75
CHAPTER 4 PAMWIN-3 ADVANCED LEVEL
4.6.1 Icon Bar
The <Icon Bar> provides commands for opening Records, viewing
comments and exporting data either as CSV data (comma separated
values) or to clipboard (compare Fig. 4.12). The functions of the
<Icon Bar> are also available in the <File> menu.
4.6.2 Sidebar
The foremost function of the <Sidebar> (Fig. 4.12) is navigation
within a <Report>. The various navigation functions for screening a
<Report> are outlines in Fig. 4.12.
4.6.3 Pick Data
The <Pick Data> panel gives access to all Saturation Pulse data and
fluorescence ratio parameters except the C/Fo data. The selection of a
parameter by ticking a checkbox results in the display of the respective data in the <Record> data window: only the selected parameters
are exported as CSV data or to the clipboard.
4.7 Script Files
Script files are used for automatic execution of experimental procedures of various complexities. Carrying out experiments by Script
files can be advantages when the same type of analysis needs to be
repeated frequently or when complicated protocols must be exactly
reproduced.
Generally, all manual operations and adjustments in the PamWin-3
software can also be carried out under the control of a Script file. In
addition, Script files offer commands for time management, combinations of sub-programs and conditional commands.
76
PAMWIN-3 ADVANCED LEVEL CHAPTER 4
To create <Script File>:
- Open <Script File> dialogue window by clicking on the Load
icon of the Script panel (sidebar of <Field Screen> or botton bar
of all <Advanced> windows.
- Click Open without selection of an existing <Script File> and
click Cancel . Enter a <Script File> name in the window which
appeared in response to the Cancel command.
Alternatively, open an existing <Script File> and click on the
Figure 4.13: Script Window.
77
CHAPTER 4 PAMWIN-3 ADVANCED LEVEL
<New Script File> (Fig. 4.13: file icons).
The <Command Box> of the <Script File> window (Fig. 4.13) contains the available commands divided into 8 sections. In addition, the
<Program Control Commands> are displayed on top of the <Command Box>. The various functions of the latter commands are comprehensibly elucidated in the <Help> attached to the <Command
Box>.
All other command groups match the commands of manual instrument operation except the group <Condition Commands) which permit execution of an instructions depending on the state of a parameter
like the fluorescence level or temperature.
A number of <Editing Tools> (Fig. 4.13) are provided to compose a
Script file. Similar as for the <Command Box>, the <Help> associated with the <Editing Tools> comprehensibly explains their function.
78
DEFINITIONS & EQUATIONS CHAPTER 5
5 Definitions and Equations
5.1 Relative Fluorescence Yields
Typically, five different types of fluorescence levels are acquired by
Saturation Pulse analyses. Two of these levels need to be established
with the dark-acclimated sample. The three remaining levels are repeatedly measured during subsequent sample treatments (e.g., exposure to Actinic Light; see Fig. 7.1).
5.1.1 Measurements with dark-acclimated samples
Fo Minimum fluorescence level excited by very low intensity of
Measuring Light to keep PS II reaction centers open.
Fm Maximum fluorescence level elicited by a strong light pulse
which closes all PS II reaction centers.
5.1.2 Measurements with light-exposed (treated) samples
Fo’ Minimum fluorescence level during a treatment. When the
measuring routine for Fo’ is active, the Fo’ level is determined during a dark interval following the Saturation Pulse.
In the dark interval, far-red light is applied to selectively
drive PS I reaction centers and, thus, to quickly remove intersystem electrons and open PS II reaction centers (see Fig.
5.1, time point 75 s).
Alternatively, the Fo’ can be estimated according to Oxborough and Baker (1997):
mF
1
Fm
1
Fo
1
1
oF
′
+−
=
′
79
CHAPTER 5 DEFINITIONS & EQUATIONS
Fm’ Maximum fluorescence levels during a treatment is induced
by saturating light pulses which temporarily close all PS II
reactions centers. Fm' is decreased with respect to Fm by
non-photochemical quenching.
F The F corresponds to the momentary fluorescence yield (Ft)
of an illuminated sample shortly before application of a Saturation Pulse.
5.2 Fluorescence Ratio Parameters
To quantify photochemical use and non-photochemical losses of absorbed light energy, fluorescence ratio expressions have been derived
which use, as data input, the relative fluorescence yield measurements introduced above. Table 5.1 compiles the fluorescence quotients available in PamWin-3. Subsequently, these fluorescence quotients will be briefly explained.
Figure 5.1: Measurements for Saturating Pulse Analysis. AL, Actinic
Light; D, dark; SP, Saturating Pulse; FR, far-red illumination.
80
DEFINITIONS & EQUATIONS CHAPTER 5
Table 5.1: Fluorescence Ratio Parameters.
Source Equation
Maximum photochemical quantum yield of
PS II (Kitajima and Butler, 1975)
F
m
FoFm
F
m
Fv
−
=
Effective photochemical quantum yield of PS II
(Genty et al., 1989)
m
F
FmF
)II(Y
′
−
′
=
Coefficient of photochemical fluorescence
quenching (Schreiber et al. 1986 as formulated
by van Kooten and Snel, 1990)
oF
m
F
FmF
q
P
′
−
′
−
′
=
Coefficient of photochemical fluorescence
quenching assuming interconnected PS II antennae (Kramer et al. 2004)
F
oF
qq
PL
′
⋅=
Coefficient of non-photochemical fluorescence
quenching (Schreiber et al. 1986 as formulated
by van Kooten and Snel, 1990)
FoF
m
oFmF
1q
N
−
′−′
−=
Stern-Volmer type non-photochemical fluorescence quenching (Bilger and Björkman, 1990)
1
mF
Fm
NPQ −
′
=
Quantum yield of non-regulated heat dissipation and fluorescence emission: this quenching
type does not require the presence of a transthylakoid ΔpH and zeaxanthin (Genty et al.
1996)*
Fm
F
)NO(Y =
Quantum yield of light-induced (ΔpH- and zeaxanthin-dependent) non-photochemical fluorescence quenching (Genty et al. 1996)*
Fm
F
mF
F
)NPQ(
Y
−
′
=
* Kramer et al. (2004) have derived more complex equations for Y(NO) and
Y(NPQ). Klughammer and Schreiber (2008) have demonstrated that the
equations by Kramer et al. (2004) can be transformed into the simple equations of (Genty et al. 1996) which are used by PamWin-3 software.
81
CHAPTER 5 DEFINITIONS & EQUATIONS
Fv/Fm and Y(II) Maximum and effective photochemical quantum
yield of PS II
Both fluorescence quotients estimate the fraction of absorbed quanta
used for PS II photochemistry, (i.e., for stable charge separation in
the PS II reaction center). For measurements of Fv/Fm, it is important
that samples are acclimated to darkness or dim light so that all reactions centers are in the open state and non-photochemical dissipation
of excitation energy is minimal. Requirements for dark acclimation
can differ between plants: in extreme shade leaves, substantial clo-
sure of PS II centers can occur already at PAR values of 0.1 μmol
photons/(m
2
·s) but many sun leaves exhibit mostly open PS II centers
even at 10-40 μmol μmol photons/(m
2
·s).
The Y(II) value estimates the photochemical use of excitation energy
in the light. To derive from the Y(II) information on the overall state
of photosynthesis, control of light conditions is required because a
leaf may be severely damaged in Calvin cycle activity and still show
a high value of Y(II) in weak light. Therefore, photosynthetic performance should be assessed during steady state illumination at a
photon flux density which is somewhat below saturation in a control
sample.
q
P
and q
L
Coefficients of photochemical fluorescence quenching.
Both parameters estimate the fraction of open PS II reaction centers.
The q
P
is based on concept of separated PS II antennae (puddle
model) but the q
L
assumes interconnected PS II antennae (lake
model) which appears to be the more realistic situation in leaves (cf.
Kramer et al., 2004). Determinations of q
P
an qL do not require fluorescence measurements with the dark-acclimated sample except when
Fo’ is not measured but calculated according to Oxborough and
Baker (1997) (see 5.1).
82
DEFINITIONS & EQUATIONS CHAPTER 5
qN and NPQ Parameters of non-photochemical quenching
Both parameters are associated with non-photochemical quenching of
excitation energy by thylakoid lumen pH- and zeaxanthin-dependent
processes. In contrast to Y(II), qP and qL, calculations of the qN and
the NPQ parameters always require fluorescence measurements with
the sample in the dark-acclimated and in the light-exposed state (see
Table 5.1). Calculation of NPQ (or SV
N
; Gilmore and Yamamoto,
1991) corresponds to the Stern-Volmer equation for fluorescence
quenching which predicts proportionality between fluorescence
quenching (NPQ) and the concentration of fluorescence-quenching
centers in the photosynthetic antennae (e.g. zeaxanthin).
Y(NO) and Y(NPQ) Yields of non-photochemical quenching
Genty et al. (1996) first presented expressions based on basic fluorescence parameters that describe the partitioning of absorbed excitation energy in PS II between three fundamental pathways, expressed
in terms of the complementary quantum yields of
Y(NO)
sum of non-regulated heat dissipation and fluorescence
emission,
Y(NPQ) regulated thermal energy dissipation involving ∆pH- and
zeaxanthin-dependent photoprotective mechanisms, and
Y(II) photochemical conversion
The yields of photochemical energy conversion and nonphotochemical losses sum up to 1:
Y(II)+Y(NPQ)+Y(NO)=1
This concept of "complementary PS II quantum yields" is useful to
analyze the partitioning of absorbed light energy in plants. For instance, under high light-conditions, a much higher Y(NPQ) than
Y(NO) indicates that excess excitation energy is safely dissipated,
83
CHAPTER 5 DEFINITIONS & EQUATIONS
that is, photosynthetic energy fluxes are well-regulated. In variance,
high values of Y(NO) would signify that excess excitation energy is
moistly channeled off via basal quenching mechanisms and, hence,
that energy fluxes are inadequately controlled.
5.3 Constant Fraction of Fo Fluorescence (C/Fo)
A number of data suggest that PS I contributes significantly to room
temperature fluorescence from leaves. Typical fractions of PS I contribution to total Fo fluorescence are 30 and 50% for C
3
and NADP-
ME C
4
plants (e.g., maize), respectively (Genty et al. 1990, Pfündel
1998, Agati et al. 2000, Peterson et al. 2001, Franck et al. 2002).
Here, the assessment of <C/Fo> is based on the comparison of the
Fo’ calculated according to Oxborough and Baker (1997) (see section
5.1.2) with the Fo’ determined during a post-Saturation Pulse illumination with far-red light. Considering that PS I fluorescence contributes a constant quantity, F
PSI
, to each fluorescence level, the Oxbor-
ough-Baker equation can be reformulated:
PSIPSIPSIPSII
PSI
FmF
1
FFm
1
FFo
1
1
FoF
−
′
+
−
−
−
+=
′
In the latter equation, the F
PSI
is subtracted from the measured fluorescence levels Fo, Fm, and Fm’ so that the total fraction in the equation above describes Fo’ originating in PS II. Clearly, the latter fraction plus the F
PSI
make up the total Fo’ signal. Compared to the origi-
nal equation, the introduction of F
PSI
increases the calculated Fo’ be-
cause the addition of F
PSI
overcompensates the effect of F
PSI
subtrac-
tion. Consequently, when the F
PSI
is disregarded, theory underestimates the Fo’, and this is consistent with the observation that the calculated Fo’ is often lower the measured Fo’. Consequently, to assess
84
DEFINITIONS & EQUATIONS CHAPTER 5
the C/Fo, the PamWin-3 program iteratively varies the level of F
PSI
until the calculated Fo’ matches the measured Fo’.
In practice, determinations of C/Fo require that the Fo and Fm and
- Fully dark-acclimated material for accurate determination of Fo
and Fm.
- Subsequent to Fo, Fm determinations, illumination with light in-
tensity that induces substantial non-photochemical quenching
but does not cause photoinhibition to measure Fo’ and Fm’.
5.4 Relative Electron Transfer Rate (ETR)
Relative electron transfer rates are calculated according to:
ETR = PAR · ETR-Factor · P
PS2/PPPS
· Y(II).
The basic idea of the ETR equation is to multiply Y(II), the effective
photochemical quantum yield of PS II, by an estimate for the photon
flux density absorbed by PS II. The latter approximation uses three
parameters which are explained below:
PAR Photosynthetically active radiation
Depending on settings, the PAR values of the active light list or
measured data are used.
ETR-Factor Absorptance of photons by photosynthetic pigments
The ETR-Factor corresponds to the fraction of incident photons absorbed by photosynthetic pigments. The default value for the ETRFactor is 0.84 which is a reasonable match to the average absorptance
in the visible range (400-700 nm) of many green leaves. Anthocyanins, however, can reduce availability of blue photons for photosynthesis (Pfündel et al. 2007) and, hence, lower the ETR-Factor.
85
CHAPTER 5 DEFINITIONS & EQUATIONS
P
PS2/PPPS
Photons absorbed by PS II relative to photons absorbed
by photosynthetic pigments.
The default value for P
PS2/PPPS
is 0.5. This value is reasonable if one
assumes only linear electron transport, that is, equal electron transfer
rates though PS I and PS II, and comparable photochemical quantum
yields of PS I and PS II under strongly light-limiting conditions.
The ETR may be compared to the rate of CO
2
-assimilation or of O2-
evolution. For such comparison the following aspects are relevant:
- 4 e- must be transported for every CO2 assimilated or O2 evolved
- the value of ETR/4 is not necessarily identical to CO
2
-fixation
rate or O
2
-evolution rate; discrepancies e. g. may arise from
photorespiratory electron flow, nitrite reduction or electron cycling at PS II
- fluorescence information primarily originates from the topmost
chloroplast layers, while gas exchange integrates over all layers;
on the other hand, the topmost layers absorb most of the light
and, hence, are also responsible for most of the gas exchange,
unless photoinhibited.
The PAR can be measured at the same spot of the leaf where fluorescence is measured, when the micro-quantum-sensor is moved into the
beam. This is possible without substantial loss in signal amplitude.
The properties of the micro-quantum-sensor are such that its response
to spectral composition and incidence angle of the impinging light
approximates that of the leaf
The combined information of ETR, PAR and Temperature provides
profound insight into the photosynthetic performance of a plant. Plots
of ETR versus PAR at different temperatures respond in a very sensitive manner to changes at all levels of the photosynthetic process (see
next section).
86
DEFINITIONS & EQUATIONS CHAPTER 5
5.5 Rapid Light Curves
The <Light Curve> feature of PamWin-3 exposes the sample to increasing intensities of Actinic illumination. Usually, the time interval
of each intensity level is too short for full equilibration of photosynthetic reactions. Therefore, these so-called “Rapid Light Curves”
(RLC) provide information on the present state of photosynthesis and
are not to be confused with classical photosynthetic light response
curves in which photosynthetic rates under steady state conditions are
plotted against light intensities. By plotting ETR versus PAR (see
previous section), Rapid Light Curves provide subsequent key parameters:
• α (alpha), electrons/photons: Initial slope of RLC which is related
to quantum efficiency of photosynthesis.
• ETR
max
, µmol electrons/(m2 · s): Maximum electron transport rate.
• I
K
, µmol photons/(m2 · s): Minimum saturating irradiance.
The function used by the PamWin-3 software has been derived by
Eilers and Peeters (1988) using a mechanistic model which considers
the processes of photosynthesis and photoinhibition. The final model
function is given next:
cPARbPARa
PAR
ETR
2
+⋅+⋅
=
To describe the data points of a Rapid Light Curve, the three free parameters (a, b, and c) of the final model function are varied until the
best
fit between function and data is achieved.
With estimates for the free parameters, the cardinal points of the
Rapid Light curves are calculated according to subsequent equations:
87
CHAPTER 5 DEFINITIONS & EQUATIONS
ca2b
c
I
ca2b
1
ETR
c
1
K
max
⋅⋅+
=
⋅⋅+
=
=α
Figure 5.2 illustrates the behavior of the Eilers and Peeters model
function for 3 theoretical cases which show identical values of α but,
at high light intensities, different degrees of photoinhibition.
Figure 5.2: Three Exemplary Rapid Light Curves.
Curve Parameter
a b c α ETR
max
Ik
1
1·10
-5
1·10
-2
5 0.2 41 207
2
1·10
-6
1·10
-2
5 0.2 69 345
3
1·10
-7
1·10
-2
5 0.2 88 438
88
DEFINITIONS & EQUATIONS CHAPTER 5
5.5.1 Some Papers on Rapid Light Curves
Fouqueray M, Mouget J-L, Morant-Manceau A, Tremblin AG (2007)
Dynamics of short-term acclimation to UV radiation in marine diatoms. J Photochem Photobiol B: Biology 89: 1–8
Jassby AD, Platt T (1976) Mathematical formulation of the relationship between photosynthesis and light for phytoplankton. Limnol
Oceanogr 21: 540-547
Perkins RG, Mouget J-L, Lefebvre S, Lavaud J (2006) Light response curve methodology and possible implications in the application of chlorophyll fluorescence to benthic diatoms. Marine Biol 149:
703-712
Platt T, Gallegos CL, Harrison WG (1980) Photoinhibition of photosynthesis in natural assemblages of marine phytoplankton. J Mar Res
38: 687-701
Ralph PJ, Gademann R (2005) Rapid light curves: A powerful tool to
assess photosynthetic activity. Aquat Bot 82: 222-237
Rascher U, Liebig M, Lüttge U (2000) Evaluation of instant light-
response curves of chlorophyll fluorescence parameters obtained with
a portable chlorophyll fluorometer on site in the field. Plant Cell Environ 23: 1397-1405
Schreiber U, Gademann R, Ralph PJ, Larkum AWD (1997) Assessment of photosynthetic performance of Prochloron in Lissoclinum
patella in hospite by chlorophyll fluorescence measurements. Plant
Cell Physiol 38: 945-951
Serôdio J, Vieira S, Cruz S, Coelho H (2006) Rapid light-response
curves of chlorophyll fluorescence in microalgae: relationship to
steady-state light curves and non-photochemical quenching in benthic diatom-dominated assemblages. Photosynth Res 90: 29-43
89
CHAPTER 5 DEFINITIONS & EQUATIONS
Serôdio J, Vieira1 S, Cruz S, Barroso F (2005) Short-term variability
in the photosynthetic activity of microphytobenthos as detected by
measuring rapid light curves using variable fluorescence. Marine Biol
146: 903-914
White AJ, Critchley C (2005) Rapid light curves: A new fluorescence
method to assess the state of the photosynthetic. Photosynth Res 59:
63-72
5.6 Literature Cited in Chapter 5
Agati G, Cerovic ZG, Moya I (2000) The effect of decreasing temperature up to chilling values on the in vivo F685/F735 chlorophyll
fluorescence ratio in Phaseolus vulgaris and Pisum sativum: the role
of the Photosystem I contribution to the 735 nm fluorescence band,
Photochem Photobiol 72: 75–84
Bilger W, Björkman O (1990) Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbance
changes, fluorescence and photosynthesis in leaves of Hedera ca-
nariensis. Photosynth Res 25:173-185
Eilers PHC, Peeters JCH (1988) A model for the relationship between light intensity and the rate of photosynthesis in phytoplankton.
Ecol Model 42: 199-215
Franck F, Juneau P, Popovic R (2002) Resolution of the Photosystem
I and Photosystem II contributions to chlorophyll fluorescence of intact leaves at room temperature. Biochim Biophys Acta 1556: 239–
246
Genty B, Briantais J-M, Baker NR (1989) The relationship between
the quantum yield of photosynthetic electron transport and quenching
of chlorophyll fluorescence. Biochim Biophys Acta 990: 87–92
90
DEFINITIONS & EQUATIONS CHAPTER 5
Genty B, Harbinson J, Cailly AL and Rizza F (1996) Fate of excitation at PS II in leaves: the non-photochemical side. Presented at The
Third BBSRC Robert Hill Symposium on Photosynthesis, March 31
to April 3, 1996, University of Sheffield, Department of Molecular
Biology and Biotechnology, Western Bank, Sheffield, UK, Abstract
P28
Genty B, Wonders J and Baker NR (1990) Non-photochemical
quenching of F0 in leaves is emission wavelength dependent: Consequences for quenching analysis and its interpretation. Photosynth Res
26: 133–139
Gilmore AM, Yamamoto HY (1991) Zeaxanthin formation and energy-dependent fluorescence quenching in pea chloroplasts under artificially mediated linear and cyclic electron transport. Plant Physiol
96: 635–643
Kitajima M, Butler WL (1975) Quenching of chlorophyll fluorescence and primary photochemistry in chloroplasts by dibromothymoquinone. Biochim Biophys Acta 376:105-115
Klughammer C and Schreiber U (2008) Complementary PS II quantum yields calculated from simple fluorescence parameters measured
by PAM fluorometry and the Saturation Pulse method. PAM Application Notes 1: 27-35
(
http://www.walz.com/e_journal/pdfs/PAN078007.pdf)
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6 Some Reviews on Chlorophyll Fluores-
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TECHNICAL SPECIFICATION CHAPTER 7
7 Specifications
7.1 Basic System
7.1.1 General Design
Battery: Built-in 12 V 2 Ah sealed Pb-gel battery
Chassis: 23 cm x 10.5 cm x 10.5 cm (L x W x H) aluminum housing
Communication: USB. Bluetooth Version 2.0 + EDR Class 2
Operating humidity range: 20 to 95% RH (avoid condensation)
Operating temperature: -5 to +40 °C
Power consumption: Basic operation 1.6 W, 8 W with all internal
light sources operated at maximum output (measuring light, red and
blue actinic light, and far red light). Saturation Pulse at maximum intensity, 30 W
Recharging time: approximately 6 hours (with the PAM-2500
turned off) via Battery Charger MINI-PAM/L
Signal detection: PIN-photodiode protected by long-pass filter
(T(50%)=715 nm). Selective window amplifier
Sockets: Connector for Special Fiberoptics 2010-F. USB socket.
Sockets for Leaf Clip Holder 2030-B or Micro Quantum/Temperature-Sensor 2060-M, Battery Charger MINI-PAM/L or
external 12 V battery via MINI-PAM/AK cable, and external
lamp/trigger output
Weight: 2.5 kg (including battery)
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CHAPTER 7 TECHNICAL SPECIFICATIONS
7.1.2 Light sources
Blue actinic light: LEDs, maximum emission at 455 nm, FWHM 20
nm, PAR up to 800 µmol/(m
2
·s), 20 intensity levels
Far-red light: LED, maximum emission at 750 nm, FWHM 25 nm,
20 intensity levels
Measuring light: Red LEDs, maximum emission at 630 nm, FWHM
(full width at half maximum) 20 nm. 1 µs pulses at modulation frequencies 10 to 5000 Hz for Fo determinations (200 Hz default), and 1
to 100 kHz during actinic illumination, fast kinetics with 100 or 200
kHz, 20 intensity levels, frequency dependent effective PAR ranging
from 0.001 to 100 µmol/(m
2
·s)
Multiple turn-over flashes: Red LEDs (see red actinic light), PAR
up to 25 000 µmol/(m
2
·s), adjustable between 1 and 300 ms, 20 in-
tensity levels
Red actinic light: LEDs, maximum emission at 630 nm, FWHM 15
nm, PAR up to 4000 µmol/(m
2
·s), 20 intensity levels
Saturation pulses: Red LEDs (see red actinic light), PAR up to
25 000 µmol/(m
2
·s), adjustable between 0.1 and 0.8 s, 20 intensity
levels
Single turn-over flashes: Red LEDs (see red actinic light), PAR up
to 125 000 µmol/(m
2
·s), adjustable between 5 and 50 µs
7.1.3 Special Fiberoptics 2010-F
Design: Flexible, steel-spiral, plastic-covered bundle with three-
pin optical connector
Joint end (measuring site): Active diameter 6 mm, outer diameter 8
mm
96
TECHNICAL SPECIFICATION CHAPTER 7
Length: 100 cm
Weight: 300 g
7.1.4 Distance Clip 60° 2010-A
Design: Metal clip with fiber holder and 11 mm sample hole: 5.5 cm
x 1.4 cm (L x W)
Fiber holder: 1.2 cm length, mounted 0.7 cm above base, with lat-
eral screw to fix fiber optics. Angle between fiber optics axis and
sample plane: 60°. Two spacer rings to vary the distance
between
fiber end and leaf surface
7.1.5 Battery Charger MINI-PAM/L
Dimensions: 15 cm x 6 cm x 3 cm (L x W x H)
Input: 90 to 264 V AC, 47 to 63 Hz
Operating temperature: 0 to 40 °C
Output: 19 V DC, 3.7 A
Weight: 300 g
7.1.6 External Voltage Supply Cable
MINI-PAM/AK cable: Bypasses the internal battery of the PAM-
2500 fluorometer and directly supplies voltage from an external 12 V
battery
7.1.7 System Control and Data Acquisition
Fluorescence parameters
97
CHAPTER 7 TECHNICAL SPECIFICATIONS
Measured: Ft, Fo, Fm, F, Fo' (also calculated), Fm'. Fast polyphasic
rise and decay kinetics (time resolution up to 10 µs). PAR and °C,
using the “Leaf-Clip Holder 2030-B” or the “Micro Quantum/Temp.Sensor 2060-M)”
Calculated: Fo' (also measured), Fv/Fm and Y(II) (maximum and effective photochemical yield of PS II, respectively), qL, qP, qN, NPQ,
Y(NPQ), Y(NO) and ETR (electron transport rate), C/Fo (constant
fraction of Fo that is assumed to originate from PS I)
Software: PamWin-3 System Control and Data Acquisition Program
7.1.8 Carrier Bag
Carrier: Robust field carrier bag with shoulder and hip belt
7.1.9 Transport Box 2040-T
Design: Aluminum box with custom foam packing for PAM-2500
and accessories
Dimensions: 60 cm x 40 cm x 25 cm (L x W x H)
Weight: 5 kg
7.1.10 Minimum Computer Requirements
Processor, 0.8 GHz. RAM, 512 MB. Hard disc space, 50 MB. Screen
resolution, 1024 x 600 pixels. Interface, USB 1.1 or USB 2.0. Optional interface, Bluetooth Version 2.0 + EDR, Widcomm Stack. Operating system: Microsoft Windows XP SP2/Vista
98
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