Photon Systems Instruments AquaPen-C AP 110-C, AquaPen-P AP 110-P Manual And User Manual

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AquaPen-C AP 110-C AquaPen-P AP 110-P

Manual and User Guide

Please read this manual before operating this product

PSI, spol. s r. o., Drásov 470, 664 24 Drásov, Czech Republic FAX: +420 511 440 901, TEL: +420 511 440 011, www.psi.cz

This document and its parts can be copied or provided to a third party only with the express permission of PSI.

The contents of this manual have been verified to correspond to the specifications of the device. However, deviations cannot be ruled out. Therefore, a complete correspondence between the manual and the real device cannot be guaranteed. The information in this manual is regularly checked, and corrections may be made in subsequent versions.

The visualizations shown in this manual are only illustrative.

This manual is an integral part of the purchase and delivery of equipment and its accessories and both Parties must abide by it.

TABLE OF CONTENTS

1 Information Before Using AquaPen Device .................................................................................................................... 5

2 Technical Specification .................................................................................................................................................... 6

3 General Information ....................................................................................................................................................... 8

3.1 Device Description ................................................................................................................................................. 9

4 List of Equipment and Customer Information .............................................................................................................. 10

5 Care and Maintenance .................................................................................................................................................. 11

6 Principle of Measurement ............................................................................................................................................ 12

7 Getting Started .............................................................................................................................................................. 15

7.1 Measurements Based on Fluorescence ............................................................................................................... 15

7.1.1 Pulses Description and Setting ........................................................................................................................ 15

7.1.2 Measurement .................................................................................................................................................. 18

7.1.3 OJIP Protocol ................................................................................................................................................... 20

7.1.4 Non-Photochemical Quenching (NPQ) Protocol ............................................................................................. 21

7.1.5 Light Curve (LC) Protocol ................................................................................................................................. 24

7.2 Optical Density Measurement (AquaPen-C only) ................................................................................................ 27

7.2.1 Calibration ....................................................................................................................................................... 27

7.2.2 Measurement .................................................................................................................................................. 27

7.3 Multiple Measurement ....................................................................................................................................... 28

8 Control Menu Tree ........................................................................................................................................................ 29

9 USB Connection ............................................................................................................................................................ 37

10 Bluetooth Connection .............................................................................................................................................. 38

10.1 Bluetooth Pairing ................................................................................................................................................. 38

11 FluorPen Software ................................................................................................................................................... 42

11.1 Software Installation ........................................................................................................................................... 42

11.2 Menu and Icon Explanation ................................................................................................................................. 43

11.2.1 Main Menu ................................................................................................................................................. 43

11.2.2 Menu Settings ............................................................................................................................................. 44

11.2.3 Menu Online Control .................................................................................................................................. 45

11.3 Data Transfer and Visualization ........................................................................................................................... 47

11.4 Firmware Update ................................................................................................................................................. 50

12 GPS Module ............................................................................................................................................................. 53

12.1 GPS / AquaPen Operation ................................................................................................................................... 53

12.2 Data Download .................................................................................................................................................... 54

13 Warranty Terms and Conditions .............................................................................................................................. 55

14 Troubleshooting and Customer Support ................................................................................................................. 56

1 INFORMATION BEFORE USING AQUAPEN DEVICE

Read this manual carefully before operating the device. If you are not sure about something in the manual, contact the manufacturer for clarification.

By accepting the device, the customer agrees to follow the instructions in this guide.

Always follow corresponding manuals while working with the AquaPen device or doing the maintenance. It is forbidden to interfere with the hardware or software of the AquaPen device in any way without previous agreement

with the manufacturer.

The following table presents basic highlight symbols used in this manual:

Symbol

Description

Important information, read carefully.

Complementary and additional information.

Tab. 1 Used symbols.

2 TECHNICAL SPECIFICATION

Measured / Calculated Parameters:

F0, Ft, Fm, Fm‘, QY, NPQ 1, NPQ 2, NPQ 3, OJIP, LC 1, LC 2, LC 3, OD

680*, OD720

Saturating Pulse Illumination:

Adjustable from 10 to 100 % (up to 3,000 µmol.m-2.s-1)

Actinic Illumination:

Adjustable from 10 to 1,000 µmol.m-2.s-1

Measuring Illumination:

Adjustable from 0 to 100 % (up to 0.09 µmol.m-2.s-1 per pulse)

LED Emitter specification:

Blue (455 nm) and red-orange (630 nm) in Cuvette version, only Blue (455nm) in Probe version

Detector Wavelength Range:

PIN photodiode with 667 to 750 nm bandpass filters

PC Communication modules:

Bluetooth and USB

Bluetooth Module:

Transfer up to 3Mbps for distance up to 20m

Built in GPS module:

Ultra-high sensitivity down to -165dBm, high accuracy of <1.5m in 50% of trials

FluorPen Software:

Windows 7 or higher compatible**

Memory Capacity:

Up to 16 Mb

Internal Data Logging:

Up to 149,000 data points

Display:

Graphical display

Keypad:

Sealed, 2-key tactile response

Keypad Escape Time:

Turns off after 8 minutes of no use

Power Supply:

Li-Ion rechargeable battery via USB cable

Battery Life:

48 hours typical with full operation

Low Battery Detection:

Low battery indication displayed

Size:

165 mm x 65 mm x 55 mm

Weight:

290 g

Sample Holder:

4 ml cuvette (AquaPen-C AP 110-C) Submersible optical probe (AquaPen-P AP 110-P)

Operating Conditions:

Temperature: 0 to +55 ºC, 32 to 130 ºF; Relative humidity: 0 to 95 % (non-condensing)

Storage Conditions:

Temperature: -10 to +60 ºC, 14 to 140 ºF; Relative humidity: 0 to 95 % (non-condensing)

Warranty:

1 year parts and labor (see the last page of this Operation Manual for precise warranty conditions)

* Only in AquaPen-C AP 110-C. ** Windows is a registered trademark of Microsoft Corporation.

Bluetooth Module Compliance Data:

3 GENERAL INFORMATION

AquaPen (AP) is a lightweight, hand-held fluorometer intended for quick and reliable measurements of photosynthetic activity in algal, cyanobacterial or plant cell suspensions. The photosynthetic activity is derived from the chlorophyll fluorescence (ChlF) kinetics. ChlF is determined based on a Pulse Amplitude Modulated technique (PAM). For user convenience, all illumination protocols are predefined and AP offers a set of illumination protocols (more in chapter 7.1) to determined fast fluorescence kinetics known as OJIP-test as well as slow ChlF kinetics such as quenching analysis or Light response curve.

AquaPen is equipped with blue and red LED emitters (AP110-C, cuvette version) or just blue in AP110-P probe version of the device. These are optically filtered and precisely focused to deliver PAR values of up to 3,000 μmol.m-2.s-1 to measured volume. Blue excitation light (455 nm) is intended for excitation of chlorophylls and thus for measurements of algal cultures and plant cell suspensions. Red-orange excitation light (630 nm) is suitable for measurements of cyanobacteria which tend to absorb inefficiently the blue light.

AquaPen is available in two versions: AquaPen-C AP110-C and AquaPen-P AP110-P.

AquaPen-P AP 110-P is a probe version, which allows detection of chlorophyll fluorescence in liquid samples by directly

submersing the probe in the suspension medium. It is designed for laboratory measurement and for field studies (in ponds and natural bodies of water). This AquaPen version is supplied with single blue LED emitter (optionally red or white).

AquaPen-C AP 110-C is a cuvette version of the fluorometer. The sample is measured in a plastic cuvette inserted into an

optical holder with a lid. This version of the AP can also be used in laboratory conditions or field studies where samples of suspension may be obtained and placed in the AP. The AP 110-C contains a built-in turbidity meter for measurements of optical densities in addition to chlorophyll fluorescence. The AP 110-C also contains two LED emitters, blue and red.

Both AP versions have ultra- high sensitivity to chlorophyll with detections of up to 0.5 μg Chl/l – therefore natural water samples containing very low concentrations of phytoplankton can be measured.

AP can be operated as a stand-alone instrument. Measured data are sequentially stored in the internal AquaPen memory. Data transfer is via USB and Bluetooth communication. Comprehensive FluorPen 1.1 software provides data transfer routines and many additional features for data viewing in tables and graphs.

AP 110-P does not measure Optical density.

3.1 DEVICE DESCRIPTION

Fig. 1 Device description.

4 LIST OF EQUIPMENT AND CUSTOMER INFORMATION

Standard version of the AquaPen device package consists:

• AquaPen-C AP 110-C or AquaPen-P AP 110-P

• Carrying Case

• 3 pieces of 4 ml volume plastic cuvette with stopper (AquaPen-C only)

• FluorPen software and driver (on a USB flash disc)

• Operation Manual (PDF on a USB flash disc)

• USB Cable

• Other Accessories or Optional Features (according to your specific order)

For data download via USB connection the USB driver needs to be installed on the PC. It can be found on the installation disk (USB driver folder).

If any item is missing, please, contact the manufacturer. Also check the carton for any visible external damage. If any damage is found, notify the carrier and the manufacturer immediately. The carton and all packing materials should be retained for inspection by the carrier or insurer.

For customer support, please write to: support@psi.cz

5 CARE AND MAINTENANCE

AquaPen-P AP 110-P

• Never submerge the whole device in the liquid!

• only the optical tip can be submerged!

• Rinse the optical tip of the AquaPen-P in freshwater after each use.

• Inspect visually the optical window after each use. If cleaning is needed, use soapy water and soft, non-

abrasive tissue for cleaning the optical part.

• The device should not come in contact with any organic solvents, strong acids or bases.

AquaPen-C AP 110-C

• Never submerge the device in water!

• To measure samples, use a standard 4 ml volume cuvette. Fill the cuvette with 3 ml of the sample. Minimal

volume for accurate measurements is 2 ml.

• Keep the optical part clean and dry. If cleaning is needed, use soft, non-abrasive tissue.

• The device should not come in contact with any organic solvents, strong acids or bases.

Li-ion battery

• Avoid fully discharging of the battery

• Do not keep the battery at full charge for all the time

• Keeping at high temperatures shortens battery life

6 PRINCIPLE OF MEASUREMENT

AquaPen is a fluorometer adapted for measurements of chlorophyll fluorescence parameters in liquid suspensions of algae, cyanobacteria and isolated plant cells . Two version of the AquaPen are available, the cuvette version (AP-C) and the probe version (AP-P). Both versions are equipped with a blue LED emitter. The cuvette version of the AquaPen also has a red LED emitter (Error! Reference source not found., Error! Reference source not found.). These are optically filtered and precisely focused to deliver light intensities of up to 3,000 µmol.m-2.s-1. Blue excitation light (455 nm) is intended for

chlorophyll excitation, i.e., for measuring chlorophyll fluorescence in algal cultures and plant cell suspensions. Redorange excitation light (630 nm) is intended for excitation of phycobilins and is suitable for measuring in cyanobacterial cultures. The AquaPen can detect chlorophyll levels down to - 0.5 µg Chl/l. Because of this high sensitivity it can be used

for measurements of natural water samples containing low concentrations of phytoplankton.

Chlorophyll fluorescence parameters measured by both versions of the AquaPen are Ft, QY, NPQ, OJIP Analysis, Light Curve response of QY. The cuvette version of the AquaPen (AP 110-C) also measures optical density at 680 and 720 nm

To use measurements of chlorophyll fluorescence to analyze photosynthesis, researchers must distinguish between

photochemical quenching and non-photochemical quenching (heat dissipation). This is achieved by stopping

photochemistry, which allows measurements of fluorescence in the presence of non-photochemical quenching alone. To

reduce photochemical quenching to negligible levels, a high intensity, short flash of light is applied to the sample. This

transiently closes all PSII reaction centers, and prevents energy of PSII being passed to downstream electron carriers. Non-

photochemical quenching will not be affected if the flash is short. During the flash, the fluorescence reaches the high level

in the absence of any photochemical quenching, known as maximum fluorescence Fm. The efficiency of photochemical

quenching (which is a proxy of the efficiency of PSII) can be estimated by comparing Fm to the steady yield of fluorescence

in the light Ft and the yield of fluorescence in the absence of photosynthetic light F0. The efficiency of non-photochemical

quenching is altered by various internal and external factors. Alterations in heat dissipation mean changes in Fm. Heat

dissipation cannot be totally stopped, so the yield of chlorophyll fluorescence in the absence of non-photochemical

quenching cannot be measured. See picture below

Fig. 2.

Fig. 2 Chlorophyll fluorescence.

Measuring the optical density of growing cultures is a common method to quantify various important culture parameters like cell concentration, biomass production or changes in the cell morphology. The cuvette version of the AquaPen measures OD at two wavelengths 680 and 720nm.

AquaPen measures:

FT - Instantaneous Chlorophyll Fluorescence

Ft is equivalent to F0 if the sample is dark-adapted.

QY - Quantum Yield

QY is a measure of the Photosystem II efficiency. QY is equivalent to Fv/Fm in dark-adapted samples and to F

v '

m '

light-adapted samples.

OJIP - Chlorophyll Fluorescence Induction Kinetics

The OJIP curves show major changes that occur during exposure of a sample to high irradiance (see more in Chapter 0).

NPQ - Non-Photochemical Quenching

The NPQ protocol is used to quantify photochemical and non-photochemical quenching. The measurement should be performed with a dark-adapted sample. (see More in chapter 7.1.4).

LC - Light Curve

Photosystem II Quantum Yield estimated from fluorescence that is measured sequentially at several different light levels. More in chapter 7.1.5.

OD - Optical Density* at 680 nm and 720 nm. (AP-C only)

Optical density at 680 nm represents light scattering and chlorophyll absorption. Optical density at 720 nm represents light scattering that corresponds to cell density. More in chapter 7.2.

*Optical density is defined as

OD = -Log(I/Io) - where "Io" is the irradiance that is transmitted through the cuvette filled with medium without algae or cyanobacteria. This quantity must be measured as the reference. "I" is the irradiance transmitted through the cuvette with algal or cyanobacterial suspension in which the OD is measured. "Log" is the decadic logarithm of the I/Io ratio. Thus, the optical density OD=1 means that the light at the respective wavelength is attenuated by the algae or cyanobacteria 10 times relative to the reference. With OD=2, the attenuation relative to the reference is 100 times.

Fig. 3 AquaPen-C AP 110-C

Fig. 4 AquaPen-P AP 110-P

7 GETTING STARTED

For more detailed information on steps of AquaPen operation please refer to chapter 8. The device is powered by built in Li-Ion battery. Ensure that the battery if fully charged by plugging it into a PC via USB

cable or the AC outlet via USB adapter (not included) and the cable.

The AquaPen is controlled using two buttons:

• Use the MENU key to scroll through sequential menu options shown on the digital display. And to turn the

device off (hold for 3 sec)

• Use the SET key to turn the device on (hold for 3 sec) and select a menu option based on cursor (>) position.

7.1 MEASUREME NTS BASE D ON FLUORES CENCE

7.1.1 PULSES DESCRIPTION AND SETTING

Flash pulse

This function serves for setting of measuring pulses intensity. The measuring pulses are weak light pulses, which are able to induce the minimal chlorophyll fluorescence (F0 or Ft). It takes only 30 µs and the maximum intensity is 3,000 µmol.m-2.s-1. It means 30 µs * 3,000 µmol.m-2.s-1 = 0.09 µmol.m-2 per pulse is the maximal intensity of the flash pulse.

Super pulse

This function serves for setting intensity of the saturating pulse. Saturating light pulse is able to induce maximum chlorophyll fluorescence (Fm). 100% of intensity represents approximately 3,000 µmol.m-2.s-1.

Actinic pulse

This function serves for setting intensity of measuring pulses. Actinic light is the ambient light in which the algae are growing. 100% of intensity equals approximately 1,000 µmol.m-2.s-1.

Pulses used in predefined protocols:

Measurements based on fluorescence

Used pulses

Flash pulse

Flash pulse, Super pulse

OJIP

Super pulse

NPQ protocols

Flash pulse, Super pulse, Actinic pulse

Light Curves

Flash pulse, Super pulse (Actinic pulse is preset)

Default setting of light color and intensities in AquaPen firmware. These may be changed according to user requirements and algal growth conditions:

Measuring color - 455nm Flash pulse 30% = Measuring flash pulse Super pulse 70% = Saturating pulse Actinic pulse 300 µmol.m-2.s-1 (30 %) = Actinic light

Please note that those paramets are recommended by manufacturer and can be change according to user needs.

Setting of optimal intensities of pulses:

Flash pulse setting

The optimum value of Flash pulse can be identified during QY measurement as shown in Fig. 5 below. Before performing QY measurement it is recommended to set the pulse color according to culture used (blue for algae and red for cyanobacteria) and intensity of Super pulse to 70 %.

Please note that QY measurement should be performed with dark adapted suspension. following the first exposure to flash pulse (during QY measurement) the sample may be inhibited and it is recommended to use a new dark adapted sample for future measurements or allow sufficient time to re-adapt the sample in the dark.

F0 increases linearly with growing intensity of the Flash pulse. The Flash pulse setting recommended by manufacturer is 30%. This intensity of Flash pulse may be increased if the culture

is very dilute. Please note that high intensities of Flash pulse can cause undesirable “actinic effect” as a result of initiated photochemistry. These effects may lower F0 and the QY values.

The optimal Flash pulse intensity is that at which the highest value of QY is reached. This can be determined by measuring QY at different flash pulse intensities using fresh dark-adapted suspensions of the same culture (Fig. 5). In this example the optimal flash pulse setting is 30%.

Fig. 5 QY measurement performed with different intensities of Flash pulse. Optimal setting is highlighted in red rectangle.

Super pulse setting

To determine the optimal intensity of the Super pulse is to perform OJIP measurement with different suspensions of the same culture at different Super Pulse settings.

Please note that OJIP measurement should be performed with dark adapted culture. Similarly as for QY measurements, new sample should be used for subsequent measurements of OJIP or sufficient time should be allowed for the sample to be dark adapted again.

The Super pulse setting recommended by manufacturer is 80 %. When performing the OJIP measurement with different intensities of Super pulse the value of Fv/Fm will stop increasing

with subsequent increases in Super pulse intensities. When that occurs the Super pulse intensity is optimal for the culture (Fig. 6 and Fig. 7).

Fig. 6 OJIP curves - measurement performed with different intensities of Super pulse.

Fig. 7 OJIP data - measurement performed with different intensities of Super pulse. The highest Fv/Fm value indicates the optimal

intensity of Super pulse (20% in this case).

Actinic pulse setting

Intensity of Actinic pulse should correspond with cultivation light intensity or should be set according to application.

If Overflow is observed on display during measurement there are two options to resolve this problem - to dilute the sample or lower the intensity of pulse.

In case of Low value on display during measurement there are two options_- concentrate the sample or increase the intensity of pulse.

7.1.2 MEASUREMENT

No device calibration is required before making chlorophyll fluorescence measurement. Results of fluorescence measurement are affected by the device settings and the physiology of the sample.

Steps for Chlorophyll Fluorescence measurements with AquaPen-C:

• Fill the cuvette with the sample of algae or cyanobacteria and close the cuvette with the stopper. Fill the

cuvette with 3 ml of the sample. Minimal volume for accurate measurements is 2 ml.

• Place the cuvette with the sample inside the AquaPen cuvette holder and close the cover to allow dark

adaptation.

• Dark adaptation of the sample is required prior to the following measurements: F0, QY, NPQ, LC). Duration

of dark-adaptation period depends on species but mostly varies between 5 and 15 minutes.

• Mix the sample to avoid sedimentation by holding the AquaPen cover and turning over a few times. This is

essential to prevent inaccurate readings

• Turn ON the device – hold SET button for 1 sec.

• Select Measurement and press SET > select required parameter for example - Ft.

• Press SET to start the measurement.

• AquaPen will measure the parameter. If a protocol was selected such as OJIP, LC or NPQ the display will only

show the progress of the measurements in % but no data will be visible.

• When measuring Ft and QY the value of the parameter will appear on the display after completion of the

measurement. To visualize the data obtained with OJIP, NPQ or LC protocol recorded data has to be download from the AquaPen to the PC computer via USB cable or the Bluetooth connection using FluorPen Software. (page 47).

• All measured data are stored in the device memory and can be downloaded to PC after completion of the

experiment.

Steps for Chlorophyll Fluorescence measurement with AquaPen-P:

• For measurements of Ft, QY, NPQ, LC the sample requires dark adaptation period of 5-15 min (this varies with

species). Place the sample in the dark to achieve this. Turn ON the device – hold SET button for 1 sec.

• Select Measurement and press SET> select required parameter for example - Ft.

• Submerge the probe in the sample and ensure that no air bubbles get trapped inside the probe.

• Press SET to start measurements.

• AquaPen will measure the parameter. If a protocol was selected such as OJIP, LC or NPQ the display will only

show the progress of the measurements in % but no data will be visible.

• When measuring Ft and QY the value of the parameter will appear on the display after completion of the

measurement. To visualize the data obtained with OJIP, NPQ or LC protocol, recorded data has to be download from the AquaPen to the PC computer via USB cable or the Bluetooth connection using FluorPen Software. (page 47).

• All measured data are stored in the device memory and can be downloaded to PC after completion of the

experiment.

7.1.3 OJIP PROTOCOL

The AquaPen device offers the protocol to capture rapid fluorescence transient – OJIP, which occurs during exposure of photosynthetic organisms to high irradiance. The FluorPen software enables data downloading to a PC and subsequent OJIP visualization of the analyzed data in a graphical and tabular format.

The OJIP protocol includes the following measured and calculated parameters:

Abbreviation

Explanation

Bckg

Background

F0 = F

50µs

, fluorescence intensity at 50 µs

Fj = fluorescence intensity at J-step (at 2 ms)

Fi = fluorescence intensity at i-step (at 30 ms)

Fm = maximal fluorescence intensity

Fv = Fm - F0 (maximal variable fluorescence)

Vj = ( Fj - F0 ) / ( Fm - F0 )

Vi = ( Fi - F0 ) / ( Fm - F0 )

Fm / F0

FV / F0

Fv / Fm

M0 or (dV/dt)0

M0 = TR0 / RC - ET0 / RC = 4 ( F

300

- F0 ) / ( Fm - F0 )

Area

Area between fluorescence curve and Fm (background subtracted)

Fix Area

Area below the fluorescence curve between F

40µs

and F

(background subtracted)

SM = Area / ( Fm - F0 ) (multiple turn-over)

SS = the smallest SM turn-over (single turn-over)

N = SM . M0 . ( 1 / VJ ) turn-over number QA

Phi_P0

Phi_P0 = 1 – ( F0 / Fm ) (or Fv / Fm)

Psi_0

Psi_0 = 1 - VJ

Phi_E0

Phi_E0 = ( 1 – ( F0 / F

)) . Psi_0

Phi_D0

Phi_D0 = 1 – Phi_P0 – ( F0 / Fm )

Phi_Pav

Phi_Pav = Phi_P0 ( SM / tFm ) t

= time to reach Fm (in ms)

ABS / RC

ABS / RC = M0 . ( 1 / VJ ) . (1 / Phi_P0 )

TR0 / RC

TR0 / RC = M0 . ( 1 / VJ )

ET0 / RC

ET0 / RC = M0 . ( 1 / VJ ) . Psi_0

DI0 / RC

DI0 / RC = ( ABS / RC ) – ( TR0 / RC )

Formulas Derived From: R.J. Strasser, A. Srivastava and M. Tsimilli-Michael (2000): The fluorescence transient as a tool to characterize and screen photosynthetic samples. In: Probing Photosynthesis: Mechanism, Regulation and Adaptation (M. Yunus, U. Pathre and P. Mohanty, eds.), Taylor and Francis, UK, Chapter 25, pp 445-483.

7.1.4 NON-PHOTOCHEMICAL QUENCHING (NPQ) PROTOCOLS

The NPQ protocol is used to quantify photochemical and non-photochemical quenching. It should be performed with darkadapted samples. The NPQ protocol starts with a measurement of minimal level of fluorescence F0 during a dark period. A short saturating flash of light is then applied to reduce the plastoquinone pool and measure maximum fluorescence in the dark-adapted state, Fm. After a short dark relaxation, the sample is exposed to actinic irradiance for tens to hundreds of seconds to elicit a transient called the Kautsky effect. A sequence of saturating flashes is then applied during the exposure to the actinic light to probe the non-photochemical quenching NPQ and effective quantum yield of photosynthesis QY in light adapted state. After exposure to continuous illumination, the relaxation of non-photochemical quenching is determined by means of saturating pulses applied in dark. This sequence of the protocol is illustrated in Fig. 8.

The AquaPen comes with three predefined NPQ protocols, NPQ1, NPQ2 and NPQ3. The protocols differ in the duration of the light exposure and the dark recovery phase, in the number and interval between pulses. See Table. 2:

Phase

Duration

# of pulses

1st pulse

Pulse interval

NPQ1

Light

60 s 5 7 s

12 s

Dark recovery

88 s 3 11 s

26 s

NPQ2

Light

200 s

10 s

20 s

Dark recovery

390 s 7 20 s

60 s

NPQ3

Light

200 s

11 s

21 s

Dark recovery

60 s 2 20 s

21 s

Table. 2 NPQ Protocols.

The NPQ protocols include the following measured and calculated parameters:

Abbreviation

Explanation

minimum fluorescence in dark-adapted state

maximum fluorescence in dark-adapted state, measured during the first saturation flash after dark adaptation

fluorescence in the peak of fast Kautsky induction

Fm_L, Lss, D, Dss

maximum fluorescence

QYmax

maximum quantum yield of PSII in dark-adapted state - Fv/F

QY_L, Lss, D, Dss

1,3

effective quantum yield of PSII

NPQ_L, Lss, D, Dss

1,4

non-photochemical chlorophyll fluorescence quenching

Qp_L, Lss, D, Dss

1,5

coefficient of photochemical quenching, an estimate of open PSII reaction centers

L - indicates light adapted parameters; D - refers to dark recovery phase after switching of the actinic illumination; n -

represents a sequential number of light phase; ss - steady state

Calculated as (Fm – F0) / Fm

Calculated as (Fm_Ln – Ft_Ln) / Fm_Ln or of corresponding steady state or dark recovery parameters

Calculated as (Fm – Fm_Ln) / Fm_Ln or of corresponding ss, Dn or Dss parameters

Calculated as (Fm_Ln – Ft_Ln) / (Fm_Ln – F0_Ln) or of corresponding ss, Dn or Dss parameters

F0_Ln is calculated as F0 / ((Fm – F0) / Fm + F0 / Fm_Ln).

For more details please refer to: Oxborough K., Baker N.R. (1997): Resolving chlorophyll a fluorescence images of photosynthetic efficiency into photochemical and non-photochemical components: calculation of qP and Fv’/Fm’ without measuring F0’. Photosynthesis Research 54: 135-142.

Fig. 8 NPQ Protocol.

7.1.5 LIGHT CURVE (LC) PROTOCOLS

The protocols called Light Curve (LC) were designed to acquire parameters for construction of Light Response Curve relating the rate of photosynthesis to photon flux density. The method is based on successive measurements of the sample exposed to a stepwise increase of light intensity. The effective quantum yields of photosynthesis are determined under various light intensities of continuous illumination. Measurement is based on pulse modulated fluorometry (PAM).

Three predefined LC protocols are available. These differ in number and duration of individual light phases and light intensities as shown in Table 3 below. The visual representation of the LC1 and LC2 protocols is shown in Figs. 8 and 9.

# of phases

Phase duration

Light intensities [µmol.m-2.s-1]

LC1

60s

10; 20; 50; 100; 300; 500

LC2

30s

100; 200; 300; 500; 1000

LC3

60s

10; 20; 50; 100; 300; 500; 1000

Table. 3 LC Protocols.

The LC protocols include the following measured and calculated parameters:

Abbreviation

Explanation

minimum fluorescence in dark-adapted state

maximum fluorescence in dark-adapted state

Fm_Ln

‡

maximum fluorescence in light adaptation state

Ft_Ln

‡

instantaneous fluorescence during light adaptation

QYmax*

maximum quantum yield of PSII in dark-adapted state - Fv/Fm

QY_Ln‡**

instantaneous PSII quantum yield induced in light

‡

n represents a sequential number of light phase *Calculated as (Fm – F0) / Fm

** Calculated as (Fm_Lx – Ft_Lx) / Fm_Lx

Fig. 9 LC1 Protocol.

Fig. 10 LC2 Protocol.

7.2 OPTICAL DENSITY MEASUREMENT (AQUAPEN -C ONLY)

7.2.1 CALIBRATION

Calibration of the AquaPen is required before measuring OD. It can be done with either plain water or ideally, culture medium placed in a cuvette and the AquaPen. Calibration assures accurate OD measurements and it is recomended before every set of samples.

It is highly recomended to calibrate AquaPen for OD measurements every time the instrument is switched ON.

Steps for calibration of the AquaPen for OD measurements:

• Use the standard 4 ml cuvette.

• Clean the cuvette with distilled water and paper tissue.

• As a calibration standard use cultivation medium (BBM, BG11 etc.) or distilled water.

• Put cuvette with medium (optimal volume 3 ml) into the AquaPen cuvette holder.

• Turn ON the device – hold SET button for 1 sec.

• select Measurement > OD > Calibration.

• Press SET button to start the calibration.

• To check the validity of the calibration select Measurement > OD > OD680nm (or OD720nm) with the blank

cuvette in the AquaPen.

• Press SET to do the measurement.

• The display should show value of 0.000.

• If the OD value is different than 0.000 repeat OD calibration again.

• Remember that the calibration is specific to a particular cuvette. New calibration should be performed for a

new cuvette.

• Calibration is automatically stored in the device memory and is saved until the device is turn OFF.

Please remember or mark the orientation of the cuvette when placed in the device. For repeated measurements it is recommended to position the cuvette always in the same orientation in the AP cuvette holder.

7.2.2 MEASUREMENT

• Fill the cuvette with a sample of algae or cyanobacteria and close the cuvette with the stopper. Minimal

volume of sample is 2 ml.

• Place the cuvette with sample inside the AquaPen cuvette holder.

• Close the cover.

• Turn the device on by pressing SET key for 1 sec

• Select from the menu Measurement > OD680nm or OD720nm.

• Press SET to start the measurement.

• Value of the measured parameter will appear on the display. All measured data are stored in the AquaPen

memory and can be downloaded to a PC via USB connection or Bluetooth connection using FluorPen software (page 47).

7.3 MULTIPLE MEASUREMENT

In addition to single measurement with each available protocol it is possible to perform multiple measurements of the same protocol over a period of time.

The AquaPen may be set up to perform repeated measurements of the same parameter or protocol by selecting in

Settings > Multi appropriate paramter or protocol (see Menu tree, page 29)

Multi type – select the required parameter - Ft, QY, OD....

Multi interval – set the time interval between measurements Multi repeats – set the number of repeated measurements Use averaging – result of some protocols can be calculated as average of multiple measurements– select YES or NO.

Recommended primarily for Ft protocol.

• Prepare the sample for measurement as described above.

• Select Measurement > Multi.

• Press SET to start.

• Values of measured parameter will appear on the AquaPen display after each repeat of measurement and

will automatically be stored to the device memory. If protocol (OJIP, NPQ, LC) was used all data will be saved to the device memory and visualization will be possible after the download of the data to the PC (page 47).

Modes of Multiple measurement:

There are two modes of multiple measurements with the AquaPen.

1. The device is connected via USB to the computer.

The device performs predefined number of measurememts and does not swicht off between measurements. Progress of the measurement is displayed in percentage in the software on the computer.

1. AquaPen is not connected to the computer

The device measures continuously according to predefined protocol and interval. The device turns on and off between measurements automatically. The multiple measurement is interrupted only by manual switching ON of the device.

8 CONTROL MENU TREE

The next few pages of this manual show the structure of the firmware menu on the AquaPen device, and explain in a schematic way the operation of the AquaPen. The schematic shows the Main Menu, first-level Sub-Menus and secondlevel Sub-Menus.

• The blue color represents the Main Menu and its Options.

• The yellow color represents the first-level Sub-Menus and their Options.

• The green color represents the second-level Sub-Menus and their Options.

• Full-line arrows are used to indicate the SET key operations.

• Dashed-line arrows are used to indicate the MENU key operations.

9 USB CONNECTION

AquaPen comes with the USB cable that is required for charging of the Li-ion battery and can also be used for data transfer to the PC after completion of measurements. To connect the USB cable with the AquaPen device Follow the picture instructions below. Please note that a lock in system is used to secure the USB cable to the AquaPen and extreme caution has to be used when setting up this connection to avoid damage to the cable pins

When connecting the USB cable take extra caution to prevent damage to the cable connector pins. Ensure correct orientation of the cable as shown in the pictures below so the circled portion of the plug and the cable in photo A and B are perfectly lined up prior to pushing them together. Once this connection is achieved the cable may be secured in position by turning the metal cover of the cable and locking the cable in position.

To connect AquaPen with your computer please follow steps below in Fig. 11:

Fig. 11 How to connect AquaPen with PC.

A) connector on the AquaPen device. B) Portion of the USB cable with pins. C – E) Position the cable horizontally and line up the green circled parts of the cable and the connector, plug in the inlet and screw the securing screw. F) Correct connection of the USB cable and Pen device.

Once the cable is securely attached to the AquaPen the other end may be connected to the USB port on a PC. The AquaPen switches ON automatically after connecting the cable to the PC. For the USB connection to be successful the USB driver and the FluorPen software need to be installed on the PC. Both may be found on the installation disk (USB driver folder) delivered with the device. Once the USB driver is installed the Device Manager in Windows will list the USB serial port in the device tree. The USB driver may also be downloaded from PSI websites www.psi.cz . Once the driver is installed correctly the connection between the AquaPen device and the computer is initiated by selecting in the software on the computer Setup > Device ID.

For more information about FluorPen software see chapter 11.

10 BLUETOOTH CONNECTION

In addition to data transfer via USB the AquarPen may be connected to the software via Bluetooth for data transfer. Before setting up the Bluetooth connection between the AquaPen and the PC, ensure the following components are in place:

1. Bluetooth enabled PC

The PC must have Bluetooth wireless technology, either built-in or through a Bluetooth card. En sure that the PC's Bluetooth setting is in "discoverable" mode (meaning that it shows up when other devices search for nearby Bluetooth connections). Consult the user guide for the PC or Bluetooth card to learn how to do this.

2. Bluetooth configuration software properly set up on the PC

Before you connecting the AquPen to the PC and downloading data files the Bluetooth software that came with the PC, or the PC's Bluetooth card needs to be activated. This software varies by manufacturer. Please consult the PC's Bluetooth documentation for more information.

3. Bluetooth must be switched on and be visible on both devices

To pair the AquaPen with another Bluetooth device, such as a computer, ensure that Bluetooth is switched on visible on both devices.

10.1 BLUETOOTH PAIRI NG

1. Enabling Bluetooth on the AquaPen

• Switch ON the AquaPen (press and hold the SET key for 1 s).

• Scroll to the Accessories menu (press the MENU key) and select Accessories by pressing the SET key.

• Select Bluetooth On (press the MENU key, then turn it ON by pressings the SET key.

Keep in mind that the AquaPen turns off automatically after about 8 minutes of no action.

Turning off the AquaPen always turns Bluetooth off.

2. Starting Bluetooth Application on the PC

The following description of how to set up the Bluetooth connection between the PC and the device is for Windows 7; some of the steps may be different with different version of Windows.

• Select: Start > Devices and Printers (Fig. 12).

You may also start your Bluetooth application via the Control Panel: Start > Control Panel > Hardware and Sound > Devices and Printers.

Fig. 12 Start Bluetooth Application.

3. Opening the Add Bluetooth Device Application

• Select: “Add a device” to start searching for the new Bluetooth device. Be sure that the AquaPen is in

discoverable mode (see step 1).

Fig. 13 Add a device.

4. Selecting the AquaPen

• Select: PSI AquaPen icon.

• Click: Next (Fig. 14).

Fig. 14 Select the AquaPen.

5. Starting the Pairing Process

This step is different for old and new version of the AquaPen, that are equipped with disparate Bluetooth module.

Old version of AquaPen (AP-100) :

Your Bluetooth Pairing Code is: 0000

• Select: “Enter the device’s pairing code”.

• Enter: 0000 (four digits).

• Select: Next (Fig. 15).

Fig. 15 Pairing process.

New version of AquaPen AP-110:

• Select: Yes (Fig. 16). Please note that the AquaPen device does not display the verification number. The

verification code is not important for the BT connection.

• Select: Next.

Fig. 16 Verifying of the BT pairing.

6. Completing the AquaPen Pairing

• Select: Close (Fig. 17).

Fig. 17 Finishing.

The Bluetooth pairing is now complete, and the next step is to open the program FluorPen 1.1 (included on the USB flash disk) For more information about FluorPen software see chapter 11.

11 FLUORPEN SOFTWARE

11.1 SOFTWARE IN STALLATION

1. Copy the FluorPen software provided on the USB flash disk to your computer and launch the FluorPen program.

2. To connect and recognize the AquaPen device in the FluorPen software, proceeds first with the registration of the

FluorPen software (Fig. 18).

• Select: Help > Register

• Enter: your serial registration number (found in a text file on the USB flash disk drive included with the device).

• Select: OK

Fig. 18 Software registration.

Please note that the serial (registration) number for the AquaPen may be found in the file SN.txt, which is included on the enclosed USB flash disk.

Please Note: it is not possible to download data from the AquaPen device without software registration.

3. Switch on the AquaPen and enable Bluetooth or connect USB cable to the PC.

4. Ensure the PC and the AquaPen are properly paired (see chapter 9 and 10 for complete information on USB and

Bluetooth pairing).

5. In the software select: Setup > Device ID (Ctrl+I). If properly connected, the message “Device: AquaPen” appears

in the bottom part of the screen (Fig. 19). If the connection is not successful then message “Device not found” will appear. In the latter case check all the connections (USB) and Bluetooth pairing

Fig. 19 Connecting AquaPen with Software.

11.2 MENU AN D IC ON EXPLANAT ION

11.2.1 MAIN MENU

MENU: File

Load Loads previously saved data files.

Save Saves data to hard disc.

Export Exports data in .txt format.

Export to JSON Exports data in JavaScript Object Notation.

Close Closes the current experiment.

Close All Closes all running experiments.

Exit Exits the program.

MENU: Device

Download Downloads data from the AquaPen to your PC.

Erase Memory Erases data from the AquaPen memory.

Online Control Online control of AP device.

Attach GPS File Used for download data from GPS module (active

only in AquaPen version AP 100).

MENU: Setup

Device ID Detects the connected device.

Update Firmware Used for firmware updates.

Settings Used for modification of the program

settings.

MENU: Help

About Offers basic information about the program.

Icon Explanation:

11.2.2 MENU SETTINGS

MENU > Setup > Settings

After Download – Memory Erase

If the box is checked the AquaPen memory is erased after each data download.

Data – Inverted

If the box is checked the polarity of data is inverted, e.g., multiplied by -1. This feature can be helpful for a certain type of experiment when the measured data are undesirably interpreted as negative values.

Data – Add to opened

If the box is checked the downloaded data are added to that of the current opened experiment.

Download

Downloads data from the AquaPen to PC.

Load

Loads (opens) previously saved data files.

Save

Saves data to hard disc.

Export

Exports data in .txt format.

Data – GPS Visible

This option is active only in older AquaPen version AP 100. In new version AP 110 the GPS data are automatically downloaded and paired with protocol measurements.

Graf – Single

If the box is checked all measured data are visualized in one graph, i.e., the value of each new measurement is added to the currently used graph window.

If the box is not checked a new graph is opened for every new measurement.

11.2.3 MENU ONLINE CONTROL This function can be used for Online Control the AquaPen device after connection with the PC.

• Select: Menu > Device > Online Control

Online Control – Switches

Sound On/Off – select presence of sound - device beeping when pressing MENU and SET keys. Measuring color – choose 455nm or 630nm for measuring fluorescence protocols. This function is active only in the

AquaPen-C 110-C model. Multi use averaging (YES/NO) – serves to confirm Repeat (number of repetitions) and Interval (time between

measurements). Settings for each measurement within Multi Option preset by the user on the AquaPen device or in the software under Values tab (see below)– select YES or NO.

Online Control – Values

One can change settings of Actinic, Super or Flash Pulse light in this window. Here is where averaging of measured parameters (Averag. count and Averag. delay) is also set up. The time between measurements (Multi-Interval) and the number of measurements (Multi-repeats) from 1-1000 can be set in this window. Finally, the type of Protocol selected (Multi-type) for Multiple measurements is also set in this window (see picture below). Please note that the Multi measurements have to be started from the device or by clicking on the Multi button in the “Protocols” tab of the Online window (see the image of the window below on pg. 47).

Online Control – Time

In this window the AquaPen time and date. One can also synchronize time of AquaPen device with computer time. This is essential for correct GPS data acquisition and therefore recommended

Online Control – Protocols

Selection of the protocol for single measurements may be done under this tab in the software. Once the measurement is completed the data is saved to the device and can be downloaded to the PC later. Also, by pressing the Multi button in this window the Multiple measurements can be started remotely. Measuring of OD 680nm and OD 720nm is active only in the AquaPen-C 110-C model.

11.3 DATA TRANSFER AND VISUALIZATION

1. Once kinetic protocols data (OJIP, NPQ, LC) have been collected with the AquaPen to visualize the data it needs

to be downloaded to the PC first via FluorPen software. Before data transfer can occur a successful connection between the AquaPen and the PC needs to be established via USB cable or Bluetooth module (see chapter 9 and 10 for details). Fig. 20 below shows example of an OJIP and NPQ protocol data.

2. Click the Download icon or select Device > Download.

3. Once the download is complete the Data can be visualized in a table shown below (Fig. 19).

Fig. 20 Example of Data Transfer and Visualization.

4. To visualize the data in the graph mode, click the Graph field in the bottom bar.

5. The selected set of data will be shown on the Graph (Fig. 21).

Fig. 21 Graphic visualization of experiment.

6. To export data from the FluorPen software select File > Export or Export icon. Select data type to export (Ft,

QY, OJIP…) - Fig. 22.

Selected only – exports only one measurement that is selected by mouse, otherwise it will export everything. Source data – exports raw data, in case of OJIP: points of the curve. Description – exports the data description if any. Computed values – export calculated data, in case of OJIP: F0, Fi, Fj...

Fig. 22 Export data.

11.4 FIRMWARE UPDATE

All data in the AquaPen memory are erased during the firmware update! Before starting any firmware update, download all your data from the AquaPen memory to the

computer!

1. Starting Update

• Select: Setup > Update Firmware From File (Fig. 23).

Fig. 23 Update Firmware.

2. Warning

• Select: OK to start update (Fig. 24)

Fig. 24 Warning.

3. Selecting .bxn file

• Find: firmware update file: Binary file (with the extension .bxn) (Fig. 25).

• Select: Open.

Fig. 25 Select .bxn file.

4. Finishing Upload

• Select: OK to start uploading of the update (Fig. 26).

Fig. 26 Data loss warning.

• The bottom bar indicates the upload progress (Fig. 27).

Fig. 27 Upload progress.

• Press: OK to finish upload (Fig. 28).

Fig. 28 Finish upload.

12 GPS MODULE

The new versions of the AquaPen device (AP110) have integrated GPS module which can be turned on during the measurements. When GPS module is turned on the map coordinates will be automatically saved with all collected data and will be downloaded during data download.

For proper GPS reading, the time in your AquaPen and in your computer must be synchronized. Preset time and time zone must correspond to GPS time (time zone) in your location.

12.1 GPS / AQUAPEN OPERATION

1. Check the time setting on the AquaPen device: Settings > Date & Time

2. Switch the GPS module “ON” on the AquaPen device by following these steps in the menu :

• Select: Accessories > GPS

• Press SET to confirm.

• Wait until the GPS position is found – “Starting GPS”.

• The GPS module is ready when the icon in upper panel changes as shown on Fig. 29.

Fig. 29 GPS icons.

3. If the picture on the display of the device does not change then proceed to Accessories>GPS>Location selection

in the menu and manually map the GPS by pressing SET. “GPS Acquisition“ message will appear followed by coordinate. If the GPS module has difficulties mapping the coordinates, a message stating “GPS not locked” will appear on the display. It may be necessary to take the device outside into a location that is easily accessible by the satellite (clear sky view) and repeat the process of mapping.

4. Once the GPS has been turned on and successfully activated proceed to Measurement and select required

protocol.

The device may need a clear view of the sky to acquire satellite signal.

Keep in mind that the AquaPen turns off automatically after about 8 minutes of no action.

Turning off the AquaPen always turns off GPS module.

For prompt determination of the coordinates use the option Accessories > GPS > Location.

12.2 DATA DOWNLOAD

1. Enabling Communication:

• Switch on the computer and the AquaPen. Set your computer to AquaPen communication: enable Bluetooth

or connect to USB port (see instructions on pg. 37).

2. Downloading Data from the AquaPen

• Start FluorPen program.

• Connect AquaPen device: Setup > Device ID (Ctrl+I)

• Download measured data from the AquaPen to your PC by clicking the down lad icon (top icon). Data

measured with activated GPS module are downloaded with GPS coordinates (Fig. 30).

Fig. 30 GPS coordinates.

13 WARRANTY TERMS AND CONDITIONS

• This Limited Warranty applies only to the AquaPen device. It is valid for one year from the date of shipment.

• If at any time within this warranty period the instrument does not function as warranted, return it and the

manufacturer will repair or replace it at no charge. The customer is responsible for shipping and insurance charges (for the full product value) to PSI. The manufacturer is responsible for shipping and insurance on return of the instrument to the customer.

• No warranty will apply to any instrument that has been (i) modified, altered, or repaired by persons

unauthorized by the manufacturer; (ii) subjected to misuse, negligence, or accident; (iii) connected, installed, adjusted, or used otherwise than in accordance with the instructions supplied by the manufacturer.

• The warranty is return-to-base only and does not include on-site repair charges such as labor, travel, or other

expenses associated with the repair or installation of replacement parts at the customer's site.

• The manufacturer repairs or replaces faulty instruments as quickly as possible; the maximum time is one month.

• The manufacturer will keep spare parts or their adequate substitutes for a period of at least five years.

• Returned instruments must be packaged sufficiently so as not to assume any transit damage. If damage is

caused due to insufficient packaging, the instrument will be treated as an out-of-warranty repair and charged as such.

• PSI also offers out-of-warranty repairs. These are usually returned to the customer on a cash-on-delivery basis.

• Wear & Tear Items (such as sealing, tubing, padding, etc.) are excluded from this warranty. The term Wear &

Tear denotes the damage that naturally and inevitably occurs as a result of normal use or aging even when an item is used competently and with care and proper maintenance.

14 TROUBLESHOOTING AND CUSTOMER SUPPORT

In case of problems with the AquaPen visit FAQ on our websites (http://psi.cz/support/faq) or contact customer support by email to support@psi.cz , or contact your local distributor.

Photon Systems Instruments AquaPen-C AP 110-C, AquaPen-P AP 110-P Manual And User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual