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

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

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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.

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TABLE OF CONTENTS

1 Information Before Using AquaPen Device .................................................................................................................... 6

2 Technical Specification .................................................................................................................................................... 7

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 How to Get Started ....................................................................................................................................................... 15

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

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

7.1.2 Measurement .................................................................................................................................................. 17

7.1.3 OJIP Protocol ................................................................................................................................................... 19

7.1.4 Non-Photochemical Quenching (NPQ) Protocol ............................................................................................. 20

7.1.5 Light Curve (LC) Protocol ................................................................................................................................. 23

7.2 Optical Density Measurement (AquaPen-C only) ................................................................................................ 26

7.2.1 Calibration ....................................................................................................................................................... 26

7.2.2 Measurement .................................................................................................................................................. 26

7.3 Multiple Measurement ....................................................................................................................................... 27

8 Control Menu Tree ........................................................................................................................................................ 28

9 USB Connection ............................................................................................................................................................ 36

10 Bluetooth Connection .............................................................................................................................................. 37

10.1 Bluetooth Pairing ................................................................................................................................................. 37

11 FluorPen Software ................................................................................................................................................... 41

11.1 Software Installation ........................................................................................................................................... 41

11.2 Menu and Icon Explanation ................................................................................................................................. 42

11.2.1 Main Menu ................................................................................................................................................. 42

11.2.2 Menu Settings ............................................................................................................................................. 43

11.2.3 Menu Online Control .................................................................................................................................. 44

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

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

12 GPS Module ............................................................................................................................................................. 52

12.1 GPS / AquaPen Operation ................................................................................................................................... 52

12.2 Data Download .................................................................................................................................................... 53

13 Warranty Terms and Conditions .............................................................................................................................. 54

14 Troubleshooting and Customer Support ................................................................................................................. 55

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1 INFORMATION BEFORE USING AQUAPEN DEVICE

Read this manual carefully before operating the device. If you are not sure about anything 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.

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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)

Detector Wavelength Range:

PIN photodiode with 667 to 750 nm bandpass filters

PC Communication modules:

Bluetooth and USB

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

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.

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3 GENERAL INFORMATION

AquaPen is a lightweight, hand-held fluorometer intended for quick and reliable measurements of photosynthetic activity in algal, cyanobacterial or eventually 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 comfort, 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, 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 AP 110-C and AquaPen-P AP 110-P.

AquaPen-P AP 110-P is a probe version of AquaPen, 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 also for field studies, when it’s necessary to measure in natural conditions. 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 a

AP optical head and covered by a top lid. This version is suitable as a hand-held laboratory fluoro-turbidimeter. It can be also used in field studies, in case it’s possible to sample. Thus, AP 110-C contains an inbuilt turbidimeter for measurements of optical densities in addition to chlorophyll fluorescence. Due to ultra- high sensitivity of this version – up to 0.5 μg Chl/l – the AquaPen-C can measure natural water samples containing very low concentrations of phytoplankton.

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

AP 110-P is not able to measure Optical density.

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3.1 DEVICE DESCRIPTION

Fig. 1 Device description.

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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

• 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 USB connection you need to have the USB driver installed in your PC. You find the driver 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 you find any damage, 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

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5 CARE AND MAINTENANCE

AquaPen-P AP 110-P

• Never submerge the whole device in the measured sample!

• Keep in mind that 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 deployment. If cleaning is needed, use the 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 almost full, i.e., about 3 ml of the

volume.

• 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.

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6 PRINCIPLE OF MEASUREMENT

AquaPen is a new version of the FluorPen fluorometer which can measure different parameters in solutions of algae, cyanobacteria and plant cell suspensions at very low concentrations. It is equipped with a blue and red LED emitter (Fig. 3, Fig. 4), optically filtered and precisely focused to deliver light intensities of up to 3,000 µmol.m-2.s-1 to measured suspensions. Blue excitation light (455 nm) is intended for chlorophyll excitation, i.e., for measuring chlorophyll

fluorescence in algal cultures and plant cell suspensions. Red-orange excitation light (630 nm) is intended for excitation through phycobilins and is suitable for measuring in cyanobacterial cultures. Due to high sensitivity - 0.5 µg Chl/l - the

AquaPen can measure natural water samples containing low concentrations of phytoplankton.

Parameters recorded by the AquaPen include Ft, QY, NPQ, OJIP Analysis, Light Curve, and also optical density at 680 and 720 nm (only AP 110-C).

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 researchers to measure 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 leaf. This transiently closes all PSII reaction centers, which 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 level reached 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 nonphotochemical quenching cannot be measured. See picture below

Fig. 2.

Fig. 2 Chlorophyll fluorescence.

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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. Continuous measuring of optical density is the most basic and powerful tool for providing optimal yields.

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 '

/F

m '

in

light-adapted samples.

OJIP - Chlorophyll Fluorescence Induction Kinetics

The OJIP curves enable observing 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 the most typically used measuring approach to quantify photochemical and non-photochemical quenching. The measurement should be performed with a dark-adapted sample. More in chapter 7.1.4.

LC - Light Curve

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

OD - Optical Density* at 680 nm and 720 nm.

Optical density at 680 nm represents light scattering and chlorophyll absorption. Optical density at 720 nm represents light scattering-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.

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Fig. 3 AquaPen-C AP 110-C.

Fig. 4 AquaPen-P 110-P.

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7 HOW TO GET STARTED

For more detailed information on particular steps of AquaPen operation please refer to chapter 8. The device works on Li-Ion battery or is charged via USB cable from PC or grid.

The AquaPen is controlled using two buttons:

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

• Use the SET key to select a menu option based on cursor (>) position.

7.1 MEASUREMENTS BASED ON FLUORESCENCE

7.1.1 PULSES DESCRIPTION AND SETTING

Flash pulse

This function serves for setting of measuring pulses intensity. The measuring pulses are weak 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 pulse is able to induce maximum chlorophyll fluorescence (Fm). 100 % of intensity equals approximately 3,000 µmol.m-2.s-1.

Actinic pulse

This function serves for setting intensity of measuring pulses. Actinic light is basically 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

Ft

Flash pulse

QY

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:

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 you can change them according to your needs.

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Setting of optimal intensities of pulses: Flash pulse setting

The optimum value of Flash pulse you can identify with QY measurement. Before QY measurement is recommended to set the pulse color according to measured culture and intensity of Super pulse on 70 %.

Please note that QY measurement should be performed with dark adapted culture. If you use cuvette version of AquaPen, “new” 4 ml of culture is needed for every measurement (culture can be inhibited after measurement).

F0 is increasing linearly with growing Flash pulse. Increasing Flash pulse is not affecting background value. The Flash pulse setting recommended by manufacturer is 30 %. You can increase the intensity of Flash pulse in case of

culture with very low density. Please note that high intensities of Flash pulse can cause undesirable “actinic effect”. In this case the Flash pulse initiates the photochemistry and affects F0 and the QY value will be lower.

The Flash pulse intensity at which the highest value of QY is reached is optimal for your culture (Fig. 5).

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

Super pulse setting

Best way to recognized the optimal intensity of Super pulse is perform OJIP measurement. Please note that OJIP measurement should be performed with dark adapted culture. If you use cuvette version of AquaPen,

“new” 4 ml of culture is needed for every measurement (culture can be inhibited after measurement). The Super pulse setting recommended by manufacturer is 70 %. Perform the OJIP measurement with different intensities of Super pulse. When the value of Fv/Fm stops growing during the

measurement with various Super pulse intensities, the Super pulse intensity is optimal for your culture (Fig. 6).

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

Actinic pulse setting

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

If you see Overflow on display during measurement there is two options how to get required result. You can dilute your sample or lower the intensity of pulse.

In case of Low value on display during measurement you can concentrate your sample or heighten the intensity of pulse.

7.1.2 MEASUREMENT

No device calibration is needed before chlorophyll fluorescence measurement. Results of fluorescence measurement depend on device settings.

Fluorescence measurement with AquaPen-C perform as follows:

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

volume of sample is 3 ml.

• Put cuvette with sample to AquaPen device and close the cover.

• If you want to prepare dark adapted sample (prior for F0, QY, NPQ, LC) please wait for defined period of

time. Duration of sufficient dark-adaptation depends on species, mostly varies between 5 and 15 minutes.

• Then mix up the sample in AquaPen (hold the cap and turn over a few times). You have to mix up the sample

after dark adaptation because of a risk of seating on the bottom of the cuvette. Sample could be wrong measured.

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

• Go to Measure > choose required parameter for example - Ft.

• Press SET to confirm.

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• AquaPen measure required parameter. If you choose protocol such as OJIP, LC or NPQ the display shows the

percentage of measurements.

• Value of required parameter appears on display after measuring. Result of OJIP, NPQ or LC protocol is a

graph, so all data you will see after data download using FluorPen Software. (page 47).

• During the measurement all data are stored into the device memory.

Fluorescence measurement with AquaPen-P perform as follows:

• If you want to prepare dark adapted sample (prior for Ft, QY, NPQ, LC) place the sample for defined period

on dark place. Duration of sufficient dark-adaptation depends on species, mostly varies between 5 and 15 minutes.

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

• Go to Measure > choose required parameter for example - Ft.

• Submerge the probe into the sample.

• Press SET to confirm.

• AquaPen measures required parameter. If you choose protocol such as OJIP, LC or NPQ the display shows the

percentage of measurements.

• Value of required parameter appears on display after measuring. Result of OJIP, NPQ or LC protocol is a

graph, so all data you will see after data download using FluorPen Software. (page 47).

• During the measurement all data are stored into the device memory.

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7.1.3 OJIP PROTOCOL

The AquaPen device offers the possibility to capture rapid fluorescence transient – OJIP, which occurs during exposure of photosynthesizing organisms to high irradiance. The FluorPen software enables data downloading to a personal computer and subsequent OJIP analysis.

The OJIP protocol includes the following extracted and technical parameters:

Abbreviation

Explanation

Bckg

Background

F0

F0 = F

50µs

, fluorescence intensity at 50 µs

Fj

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

Fi

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

Fm

Fm = maximal fluorescence intensity

Fv

Fv = Fm - F0 (maximal variable fluorescence)

Vj

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

Vi

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

1s

(background subtracted)

SM

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

SS

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

N

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

M

)) . Psi_0

Phi_D0

Phi_D0 = 1 – Phi_P0 – ( F0 / Fm )

Phi_Pav

Phi_Pav = Phi_P0 ( SM / tFm ) t

Fm

= 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.

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7.1.4 NON-PHOTOCHEMICAL QUENCHING (NPQ) PROTOCOL

The NPQ protocol is the most typically used measuring approach to quantify photochemical and non-photochemical quenching. The measurement should be performed with a dark-adapted sample. Thereby, it may not be appropriate under field conditions.

The NPQ protocol starts by giving a measuring light to acquire minimal level of fluorescence F0. 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 of the Kautsky effect. Moreover, a sequence of saturating flashes is applied on top of the actinic light to probe the nonphotochemical 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 (Fig. 7).

Three NPQ protocols, NPQ1, NPQ2 and NPQ3 are predefined. The protocols differ in the duration of the light exposure and the dark recovery phase, in the number and interval between pulses. See Tab. 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

10 s

20 s

Dark recovery

390 s 7 20 s

60 s

NPQ3

Light

200 s

10

11 s

21 s

Dark recovery

60 s 2 20 s

21 s

Tab. 2 NPQ Protocols.

The protocol includes following measured and calculated parameters:

Abbreviation

Explanation

F

0

minimum fluorescence in dark-adapted state

F

m

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

F

p

fluorescence in the peak of fast Kautsky induction

Fm_L, Lss, D, Dss

1

maximum fluorescence

QYmax

2

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

m

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

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1

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

2

Calculated as (Fm – F0) / Fm

3

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

4

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

5

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.

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Fig. 7 NPQ Protocol.

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7.1.5 LIGHT CURVE (LC) PROTOCOL

The protocol called Light Curve (LC) was 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).

Several LC protocols are predefined in AP. These differ in number and duration of individual light phases and light intensities.

# of phases

Phase duration

Light intensities [µmol.m-2.s-1]

LC1 6 60s

10; 20; 50; 100; 300; 500

LC2 5 30s

100; 200; 300; 500; 1000

LC3 7 60s

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

Tab. 3 LC Protocols.

The protocol includes following measured and calculated parameters:

Abbreviation

Explanation

F

0

minimum fluorescence in dark-adapted state

F

m

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

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Fig. 8 LC1 Protocol.

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Fig. 9 LC2 Protocol.

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7.2 OPTICAL DENSITY MEASUREMENT (AQUAPEN-C ONLY )

7.2.1 CALIBRATION

Blank – either water or ideally medium used to cultivate the culture you plan to measure in AquaPen. Calibration assures you that your measurements are accurate within the specification limits that led you to select the instrument in the first place. It is better to calibrate your device before every set of samples.

It is necessary to calibrate AquaPen after every switch ON.

Prior OD measurement perform the calibration as follows:

• 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 (minimal volume 2 ml) into the AquaPen device.

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

• Go to Measurement > OD > Calibration.

• Press SET button to confirm the calibration.

• For checking if the calibration went well go to Measurement > OD > OD680nm (or OD720nm).

• Press SET to confirm the measurement.

• On display should appear value 0.000.

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

• Remember that the calibration is done for the particular cuvette. If you want to use another cuvette, you

should make the calibration with this one.

• Calibration is automatically stored into the device memory and is save till the device turn OFF.

Please remember or mark the orientation of the cuvette 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.

• Put cuvette with sample to AquaPen device.

• Close the cover.

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• Go to Measurement > OD680nm or OD720nm.

• Press SET to confirm.

• Value of required parameter appears on display after measuring. Measured data are stored into the device

memory and can be downloaded to PC (page 47).

7.3 MULTIPLE MEASUREMENT

Except single measurement you can perform multiple measurements. In this mode AquaPen repeats measurements according to parameters, which you have set in Settings > Multi (see Menu

tree, page 28)

Multi type – choose your required parameter - Ft, QY, OD....

Multi interval – set the time interval between measurements Multi repeats – set the number of repeated measurements Use averaging – serves to confirm Repeat and Interval Options for each measurement within Multi Option – select YES or

NO.

• Prepare the sample like for single measurement.

• Go to Measurement > Multi.

• Press SET to confirm.

• Values appears on display after each repeat of measurement and are automatically stored to the device

memory. If you used protocol (OJIP, NPQ, LC) all data you will see after download data (page 47).

Modes of Multiple measurement:

1. AquaPen is connected via USB to computer

The device measures predefined number of repeats and does not swicht off between measurements. Progress of the measurement is displayed in percentage.

2. AquaPen is not connected to the computer

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

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8 CONTROL MENU TREE

The next pages show the structure of the operation scheme, which includes the Main Menu, first-level Sub-Menus and second-level 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 for the SET key.

• Dashed-line arrows are used for the MENU key.

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9 USB CONNECTION

Connect the USB cable with the AquaPen device. Please note that lock in system is used.

Pay attention when connecting the USB cable not to damage the outlet connector on the Pen device. Make sure that you orient the cable correctly prior connecting the inlet with the outlet and the cable is upright towards the device.

To connect AquaPen with your computer please follow steps below in Fig. 10:

Fig. 10 How to connect AquaPen with PC.

A) Outlet connector on AquaPen device. B) Inlet part on the USB cable. C – E) Position the cable horizontally, plug in the inlet and screw the securing screw. F) Correct connection of the USB cable and Pen device.

Connect the USB cable to a computer. The AquaPen switches ON automatically after connecting the cable to the PC. For USB connection you need to have the USB driver installed in your PC. You find the driver on the installation disk (USB driver folder). If you check the Device Manager in Windows you should see the USB serial port in the device tree. In case of missing driver, you may download it from PSI websites. When the driver is installed correctly you should be able to connect to the device in the FluorPen software menu Setup > Device ID.

For more information about FluorPen software see chapter 11.

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10 BLUETOOTH CONNECTION

Before you set up the Bluetooth connection between the AquaPen and your PC, make sure you have these components:

1. Bluetooth enabled PC

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

2. Bluetooth configuration software properly set up on PC

Before you can download files to your PC, you will need to set up the Bluetooth software that came with your PC, or your PC's Bluetooth card. This software varies by manufacturer. Please consult your PC's Bluetooth documentation for more information.

3. Bluetooth must be switched on visible on both devices

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

10.1 BLUETOOTH PAIRING

1. Enabling Bluetooth in the AquaPen

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

• Scroll to the Accessories menu (press the MENU key, then press the SET key).

• Select Bluetooth On (press the MENU key, then press the SET key) to enable Bluetooth.

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

Turning off the AquaPen always turns off Bluetooth.

2. Starting Bluetooth Application on Your PC

Be aware that this description is working on Windows 7; some of the steps may be different on your PC.

• Select: Start > Devices and Printers (Fig. 11).

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

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Fig. 11 Start Bluetooth Application.

3. Opening the Add Bluetooth Device Application

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

mode (see step 1).

Fig. 12 Add a device.

4. Selecting the AquaPen

• Select: PSI AquaPen icon.

• Select: Next (Fig. 13).

Fig. 13 Select the AquaPen.

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5. Starting the Pairing Process

This step is different for old and new version of Bluetooth module.

Old version:

Your Bluetooth Pairing Code is: 0000

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

• Enter: 0000 (four digits).

• Select: Next (Fig. 14).

Fig. 14 Pairing process.

New version:

• Select: Yes (Fig. 15). Please note that the FluorPen device does not display the verification number. The

verification code is not important for the BT connection.

• Select: Next.

Fig. 15 Verifying of the BT pairing.

6. Completing the AquaPen Pairing

• Select: Close (Fig. 16).

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Fig. 16 Finishing.

On computer run the program FluorPen 1.1. For more information about FluorPen software see chapter 11.

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11 FLUORPEN SOFTWARE

11.1 SOFTWARE INSTALLATION

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

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

your FluorPen software (Fig. 17).

• Select: Help > Register

• Enter: your serial registration number.

• Select: OK

Fig. 17 Software registration.

Please note that you will find your serial (registration) number in the file SN.txt, which is included on the enclosed USB flash disk.

Please remember: 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. Make sure that your PC and the AquaPen are properly paired (see chapter 9 and 10 for complete information on

USB and Bluetooth pairing).

5. Select: Setup > Device ID (Ctrl+I). If properly connected, the message “Device: AquaPen” appears in the bottom

part of the screen (Fig. 18).

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Fig. 18 Connecting AquaPen with Software.

11.2 MENU AND ICON EXPLANATION

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).

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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.

Register Used for the FluorPen software registration.

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.

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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 your AquaPen device after connection with your PC.

• Select: Menu > Device > Online Control

Online Control – Switches

Sound On/Off - choose On/Off for device beeping. 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) Options for each measurement within Multi Option – select YES or NO.

Online Control – Values

You can change settings of Actinic, Super or Flash Pulse light. You can also set Averaging of Ft, OJIP, NPQ, LC measurements. You can set time between measurements (Interval) and also you can set number of Multiprotocol measurements (1-1000), type of Multiprotocol or time between measurements.

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Online Control – Time

Set the AquaPen time a date. You can also synchronize time of AquaPen device with computer time.

Online Control – Protocols

Choose the protocol for measuring. AquaPen device start to measure. After measuring you can download data. Measuring of OD 680nm and OD 720nm is active only in the AquaPen-C 110-C model.

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11.3 DATA TRA NSFER AND VISUALIZAT ION

1. Perform a measurement with your AquaPen. In Fig. 19 we did an OJIP measurement.

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

3. The Data table appears.

Fig. 19 Example of Data Transfer and Visualization.

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4. To visualize measurement in the graphic mode, click the Graph field in the bottom bar.

5. The colored Graph of measured data appears (Fig. 20).

Fig. 20 Graphic visualization of experiment.

6. For export press File > Export or Export icon, select what you want to export (Ft, QY, OJIP…) - Fig. 21.

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...

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Fig. 21 Export data.

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11.4 FI RMWARE UPDATE

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

computer!

1. Starting Update

• Select: Setup > Update Firmware From File (Fig. 22).

Fig. 22 Update Firmware.

2. Warning

• Select: OK to start update (Fig. 23)

Fig. 23 Warning.

3. Selecting .bxn file

• Find: Binary file (with the extension .bxn) (Fig. 24).

• Select: Open.

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Fig. 24 Select .bxn file.

4. Finishing Upload

• Select: OK to start uploading of the update (Fig. 25).

Fig. 25 Data loss warning.

• The bottom bar indicates the upload progress (Fig. 26).

Fig. 26 Upload progress.

• Press: OK to finish upload (Fig. 27).

Fig. 27 Finish upload.

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12 GPS MODULE

AquaPen device has integrated GPS module which can be turned on during the measurement and the GPS coordinated will be added to the downloaded data.

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 in AquaPen device: Settings > Date & Time

2. Switch the GPS module on:

• 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 change – see on Fig. 28.

Fig. 28 GPS icons.

3. Go to Measurement and choose 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.

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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.

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. Data measured with activated GPS module are

downloaded with GPS coordinates (Fig. 29).

Fig. 29 GPS coordinates.

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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.

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14 TROUBLESHOOTING AND CUSTOMER SUPPORT

In case of troubles and for customer support, please, visit FAQ on our websites, write to support@psi.cz or contact your local distributor.

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

Specifications and Main Features

Frequently Asked Questions

User Manual