Photon Systems Instruments AquaPen-C AP 110-P Guide

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 .................................................................................................................... 6
2Technical Specification.................................................................................................................................................... 7
3General Information ....................................................................................................................................................... 8
3.1 Device Description................................................................................................................................................. 9
4List of Equipment and Customer Information .............................................................................................................. 10
5Care and Maintenance.................................................................................................................................................. 11
6Principle of Measurement ............................................................................................................................................ 12
7How 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
8Control Menu Tree........................................................................................................................................................ 28
9USB 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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1INFORMATION 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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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)
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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3GENERAL 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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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
•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: suppo[email protected]

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5CARE 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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6PRINCIPLE 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 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. Continuous measuring of optical density is
the most basic and powerful tool for providing optimal yields.
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 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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7HOW 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 (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 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 F0and 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/Fmstops 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= 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.

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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 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 (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
F0
minimum fluorescence in dark-adapted state
Fm
maximum fluorescence in dark-adapted state, measured during the first
saturation flash after dark adaptation
Fp
fluorescence in the peak of fast Kautsky induction
Fm_L, Lss, D, Dss1
maximum fluorescence
QYmax2
maximum quantum yield of PSII in dark-adapted state - Fv/Fm
QY_L, Lss, D, Dss1,3
effective quantum yield of PSII
NPQ_L, Lss, D, Dss1,4
non-photochemical chlorophyll fluorescence quenching
Qp_L, Lss, D, Dss1,5
coefficient of photochemical quenching, an estimate of open PSII reaction centers