Photon Systems Instruments AquaPen-C AP 110-C Manual

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Manual Version: 2021/12
© PSI (Photon Systems Instruments), spol. s r.o.
www.psi.cz
This document and its parts can be copied or provided to a third party only with the express permission of PSI.
The contents of this manual have been verified to correspond to the specifications of the device. However, deviations cannot be ruled out.
Therefore, a complete correspondence between the manual and the real device cannot be guaranteed. The information in this manual is
regularly checked, and corrections may be made in subsequent versions.
The visualizations shown in this manual are only illustrative.
This manual is an integral part of the purchase and delivery of equipment and its accessories and both Parties must abide by it.

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TABLE OF CONTENT
1Information before using AquaPen device........................................................................................................ 5
2General Description ......................................................................................................................................... 6
2.1 Technical Specification..............................................................................................................................................7
3Device Description ........................................................................................................................................... 9
3.1 List of equipment and customer information..........................................................................................................10
3.2 Care and maintenance............................................................................................................................................10
4Principle of measurement.............................................................................................................................. 11
5Getting started............................................................................................................................................... 14
5.1 Measurements based on fluorescence....................................................................................................................14
5.1.1 Pulses description and setting ................................................................................................................................14
5.1.2 Measurement .........................................................................................................................................................18
5.1.3 OJIP protocol ..........................................................................................................................................................18
5.1.4 Non-photochemical quenching (NPQ) protocols....................................................................................................20
5.1.5 Light curve (LC) protocols .......................................................................................................................................23
5.2 Optical density measurement (AquaPen-C only) ....................................................................................................26
5.2.1 Calibration ..............................................................................................................................................................26
5.2.2 Measurement .........................................................................................................................................................26
5.3 Multiple measurement ...........................................................................................................................................27
6Control menu tree.......................................................................................................................................... 28
7USB Connection ............................................................................................................................................. 36
8Bluetooth connection..................................................................................................................................... 37
8.1 Bluetooth pairing....................................................................................................................................................37
9FluorPen software.......................................................................................................................................... 40
9.1 Software installation...............................................................................................................................................40
9.2 Menu and icons explanation...................................................................................................................................41
9.2.1 Main menu .............................................................................................................................................................41
9.2.2 Menu settings.........................................................................................................................................................42
9.2.3 Menu online control ...............................................................................................................................................42
9.3 Data transfer and visualization...............................................................................................................................45
9.4 Firmware update ....................................................................................................................................................48
10 GPS module ................................................................................................................................................... 50

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10.1 GPS/AquaPen operation .........................................................................................................................................50
10.2 Data download .......................................................................................................................................................50
11 Warranty terms and conditions...................................................................................................................... 52
12 Troubleshooting and Customer Support......................................................................................................... 52

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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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2GENERAL DESCRIPTION
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 Error! Reference source not found.) to determined fast fluorescence k
inetics known as OJIP-test as well as slow ChlF kinetics such as quenching analysis or Light response curve.
AquaPen is available in two versions: AquaPen-C AP110-C and AquaPen-P AP110-P.
AquaPen AP110-C, cuvette version is equipped with blue (455 nm) and red (630 nm) LED emitters whereas AquaPen AP110-P, probe
version incorporates just blue (470 nm) LED emitter. 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 is intended for excitation of chlorophylls and thus for measurements of algal
cultures and plant cell suspensions. Red-orange excitation light is suitable for measurements of cyanobacteria which tend to absorb
inefficiently the blue light.
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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2.1 TECHNICAL SPECIFICATION
Protocols
Ft–instantaneous chlorophyll fluorescence
Quantum Yield
OJIP
Non-photochemical quenching
Light curve
Optical density at 680 and 720 nm (AP 110-C only)
LED emitter
AP 110-C: Red-orange (630 nm) and blue (455 nm)
AP 110-P: Blue (470 nm), other wavelengths on request
Saturating pulse Illumination
Up to 3,000 µmol(photon).m-2.s-1 (adjustable from 10 to 100%)
Actinic Illumination
Adjustable from 10 to 1,000 µmol(photon).m-2.s-1
Measuring Illumination
Up to 0,09 µmol(photon).m-2.s-1 per pulse (adjustable from 10 to 100%)
Detector
PIN photodiode with bandpass filters
Wavelength range from 667 to 750 nm
Internal memory capacity
Up to 16 Mb
Internal data logging
Up to 149,000 measurements (depending on protocol)
Data transfer
USB cable
Bluetooth (transfer up to 3Mbps for distance up to 20m)
PC software
FluorPen 1.1 (Windows 7 and higher)
Battery
Li-Ion rechargeable battery
Capacity 2000 mAh
Max. charging current 0.5 A
Charging via USB port - PC, power bank, USB charger, etc.
48 hours typical with full operation
Low battery indicator
Sample holder
AP 110-C: 4 ml cuvette
AP 110-P: Submersible optical probe
Display
Graphical display
Keypad
Sealed, 2-key tactile response
Turns off after 5 minutes of no use
Built in GPS module
Ultra-high sensitivity down to -165dBm
High accuracy of <1.5 m in 50% of trials
Size
165 x 65 x 55 mm
Weight
290 g
Operating conditions
Temperature: 0 to +55 °C
Relative humidity: 0 to 95 % (non-condensing)
Storage conditions
Temperature: -10 to +60 °C
Relative humidity: 0 to 95 % (non-condensing)
Warranty
1-year parts and labor

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Bluetooth module compliance data:
Category
Country
Standard
Radio
USA
FCC Part 15 Subpart B: 2008 Class B
FCC CRF Title 47 Part 15 Subpart C
FCC ID:
T9J-RN42
Europe
ETSI EN 301 489-1 V1.8.1
ETSI EN 301 489-17 V2.1.1
ETSI EN 300 328 V1.7.1
Canada
IC RSS-210 low power comm. device
Certification number:
6514A-RN42
EMC
USA
FCC CFR47 Part 15 subclass B
Europe
EN 55022 Class B radiated
EN61000-4-2 ESD immunity
EN61000-4-3 radiated field
EN61000-4-6 RF immunity
EN61000-4-8 power magnetic immunity

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3DEVICE DESCRIPTION
Fig. 1 Device description.

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3.1 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
•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: [email protected]
3.2 CARE AND MAINTENANCE
AquaPen-P AP 110-P
•Never submerge the whole device in the liquid!
•Only the optical tip can be submerged!
•Rinse the optical tip of the AquaPen-P in freshwater after each use.
•Inspect visually the optical window after each use. If cleaning is needed, use soapy water and soft, non-abrasive tissue for cleaning
the optical part.
•The device should not come in contact with any organic solvents, strong acids or bases.
AquaPen-C AP 110-C
•Never submerge the device in water!
•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.
•To measure samples, use a standard 4 ml volume cuvette (plastic cuvettes with 4 clear faces for visible range from Kartell are
recommended). Fill the cuvette with 3 ml of the sample. Minimal volume for accurate measurements is 2 ml.
•Clean the cuvettes with distilled water, avoid contact with alcohol and solvents.
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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4PRINCIPLE OF MEASUREMENT
AquaPen is a fluorometer adapted for measurements of chlorophyll fluorescence parameters in liquid suspensions of algae,
cyanobacteria and isolated plant cells. Two versions 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 (455 nm for AP-C, 470 nm for AP-P). The cuvette version of the AquaPen also
has a red LED emitter (Fig. 3). These are optically filtered and precisely focused to deliver light intensities of up to 3,000 µmol.m-2.s-1.
Blue excitation light is intended for chlorophyll excitation, i.e., for measuring chlorophyll fluorescence in algal cultures and plant cell
suspensions. Red-orange excitation light 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 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 F0 if 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 5.1.3).
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 5.1.4).
LC - Light Curve
Photosystem II Quantum Yield estimated from fluorescence that is measured sequentially at several different light levels. More in
chapter 5.1.5.
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 5.2.
*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 AP110-C.
Fig. 4 AquaPen-P AP 110-P.

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5GETTING STARTED
For more detailed information on steps of AquaPen operation please refer to chapter Error! Reference source not found..
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.
5.1 MEASUREMENTS BASED ON FLUORESCENCE
5.1.1 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 actinic light. It 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)

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Default setting of light color and intensities in AquaPen firmware. These may be changed according to user requirements and algal
growth conditions:
Measuring color –455 (470, respectively) nm
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 parameters are recommended by manufacturer and can be change according to user
needs.
Setting of optimal intensities of pulses:
Flash pulse setting
The optimum value of Flash pulse can be identified during QY measurement as shown in Fig. 5 below. Before performing QY measurement
it is recommended to set the pulse color according to culture used (blue for algae and red for cyanobacteria) and intensity of Super pulse
to 70 %.
Please note that QY measurement should be performed with dark adapted suspension. following the first exposure to flash pulse (during
QY measurement) the sample may be inhibited and it is recommended to use a new dark-adapted sample for future measurements or
allow sufficient time to re-adapt the sample in the dark.
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.

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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 (Error! Reference source not f
ound. and Error! Reference source not found.).
Fig. 6 OJIP curves - measurement performed with different intensities of Super pulse.

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Fig. 7 OJIP data - measurement performed with different intensities of Super pulse. The highest Fv/Fm value indicates the optimal intensity of
Super pulse (20% in this case).
Actinic pulse setting
Intensity of Actinic pulse should correspond with cultivation light intensity or should be set according to application.
If Overflow is observed on display during measurement there are two options to resolve this problem - to dilute
the sample or lower the pulse intensity.
In case of Low value on display during measurement there are two options - concentrate the sample or increase
the pulse intensity.

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5.1.2 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 44).
•All measured data are stored in the device memory and can be downloaded to PC after completion of the experiment.
Steps for Chlorophyll Fluorescence measurement with AquaPen-P:
•For measurements of Ft, QY, NPQ, LC the sample requires dark adaptation period of 5-15 min (this varies with species). Place the
sample in the dark to achieve this. Turn ON the device –hold SET button for 1 sec.
•Select Measurement and press SET> select required parameter for example - Ft.
•Submerge the probe in the sample and ensure that no air bubbles get trapped inside the probe.
•Press SET to start measurements.
•AquaPen will measure the parameter. If a protocol was selected such as OJIP, LC or NPQ the display will only show the progress of
the measurements in % but no data will be visible.
•When measuring Ft and QY the value of the parameter will appear on the display after completion of the measurement. To visualize
the data obtained with OJIP, NPQ or LC protocol, recorded data has to be download from the AquaPen to the PC computer via USB
cable or the Bluetooth connection using FluorPen Software (page 44).
•All measured data are stored in the device memory and can be downloaded to PC after completion of the experiment.
5.1.3 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.

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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.

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5.1.4 NON-PHOTOCHEMICAL QUENCHING (NPQ) PROTOCOLS
The NPQ protocol is used to quantify photochemical and non-photochemical quenching. It should be performed with dark-adapted
samples. The NPQ protocol starts with a measurement of minimal level of fluorescence F0during a dark period. A short saturating flash
of light is then applied to reduce the plastoquinone pool and measure maximum fluorescence in the dark-adapted state, Fm. After a short
dark relaxation, the sample is exposed to actinic irradiance for tens to hundreds of seconds to elicit a transient called the Kautsky effect.
A sequence of saturating flashes is then applied during the exposure to the actinic light to probe the non-photochemical quenching NPQ
and effective quantum yield of photosynthesis QY in light adapted state. After exposure to continuous illumination, the relaxation of
non-photochemical quenching is determined by means of saturating pulses applied in dark. This sequence of the protocol is illustrated
in Fig. 8.
The AquaPen comes with three predefined NPQ protocols, NPQ1, NPQ2 and NPQ3. The protocols differ in the duration of the light
exposure and the dark recovery phase, in the number and interval between pulses. See Table 1 NPQ Protocols.Table 1:
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
Table 1 NPQ Protocols.
The NPQ protocols include the 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_Ln, Lss, D, Dn1
maximum fluorescence
QYmax2
maximum quantum yield of PSII in dark-adapted state - Fv/Fm
QY_Ln, Lss, D, Dn1,3
effective quantum yield of PSII
NPQ_Ln, Lss, D, Dn1,4
non-photochemical chlorophyll fluorescence quenching
Qp_Ln, Lss, D, Dn1,5
coefficient of photochemical quenching, an estimate of open PSII reaction centers
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 phases; ss - steady state
2 Calculated as (Fm–F0) / Fm
3Calculated 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