Photon Systems Instruments AquaPen-C AP 110-C Guide

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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: [phone], TEL: [phone], www.psi.cz

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© PSI (Photon Systems Instruments), spol. s r. o. (hereinafter PSI), 2018
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
1Information Before Using AquaPen Device .................................................................................................................... 5
2Technical Specification.................................................................................................................................................... 6
3General Information ....................................................................................................................................................... 8
3.1 Device Description................................................................................................................................................. 9
4List of Equipment and Customer Information .............................................................................................................. 10
5Care and Maintenance.................................................................................................................................................. 11
6Principle of Measurement ............................................................................................................................................ 12
7Getting 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
8Control Menu Tree........................................................................................................................................................ 29
9USB 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

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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

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1INFORMATION 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.

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2TECHNICAL SPECIFICATION
Measured / Calculated Parameters:
F0, Ft, Fm, Fm‘, QY, NPQ 1, NPQ 2, NPQ 3, OJIP, LC 1, LC 2, LC 3, OD680*, 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)

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* Only in AquaPen-C AP 110-C.
** Windows is a registered trademark of Microsoft Corporation.
Bluetooth Module Compliance Data:

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3GENERAL 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.

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3.1 DEVICE DESCRIPTION
Fig. 1 Device description.

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4LIST 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: suppo[email protected]

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5CARE 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

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6PRINCIPLE 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. Red-
orange 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 Fmto the steady yield of fluorescence
in the light Ftand 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.

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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. The cuvette version of the AquaPen
measures OD at two wavelengths 680 and 720nm.
AquaPen measures:
FT- Instantaneous Chlorophyll Fluorescence
Ftis equivalent to F0if the sample is dark-adapted.
QY - Quantum Yield
QY is a measure of the Photosystem II efficiency. QY is equivalent to Fv/Fmin dark-adapted samples and to Fv ' /Fm ' in
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.

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Fig. 3 AquaPen-C AP 110-C
Fig. 4 AquaPen-P AP 110-P

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7GETTING 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 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 light pulses, which are able
to induce the minimal chlorophyll fluorescence (F0or 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
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 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:

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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.
F0increases 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 F0and 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/Fmwill 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).

© PSI (Photon Systems Instruments), spol. s r. o.
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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).

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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.

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•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.

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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
F0= F50µs, 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 ( F300 - F0 ) / ( Fm - F0 )
Area
Area between fluorescence curve and Fm (background subtracted)
Fix Area
Area below the fluorescence curve between F40µs and F1s (background subtracted)
SM
SM= Area / ( Fm - F0 ) (multiple turn-over)
SS
SS= the smallest SMturn-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/ FM )) . Psi_0
Phi_D0
Phi_D0= 1 –Phi_P0 –( F0/ Fm )
Phi_Pav
Phi_Pav = Phi_P0 ( SM / tFm ) tFm = 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.