Walz PHYTO-PAM-II User manual

Printed in Germany
Phytoplankton Analyzer
PHYTO-PAM-II
and
Phyto-Win 3
Software V 3
Principles of Operation
2nd Edition: Nov 2016
PhytoPamII_2.doc
Heinz Walz GmbH, 2016
Heinz Walz GmbH Eichenring 6 91090 Effeltrich Germany
Phone [phone] Telefax +49-(0)9133/5395
Email [email protected] Internet www.walz.com

MAY 2002 CONTENTS
1Safety instructions ........................................................................ 1
1.1General safety instructions .......................................................... 1
1.2Special safety instructions........................................................... 2
2PHYTO-PAM-II........................................................................... 3
2.1Compact Version......................................................................... 5
Spherical Micro Quantum Sensor US-SQS/WB.................... 82.1.1
Stirring Device WATER-S (optional)....................................92.1.2
Battery Charger MINI-PAM/L ............................................ 102.1.3
3PhytoWin Software installation and Phyto-PAM-II setup..... 11
3.1Measure PAR Lists.................................................................... 13
4First fluorescence measurements ............................................. 16
4.1Principle of distinguishing between different groups of
phytoplankton............................................................................ 21
5Saturation Pulse Analysis .......................................................... 23
5.1Measurements with Dark-Acclimated Samples ........................ 23
5.2Measurements with Illuminated Samples.................................. 24
5.3Fluorescence Ratio Parameters ................................................. 25
6Features of PhytoWin Software ................................................ 30
6.1Elements for system operation and display of instrument status33
6.2Channels-window...................................................................... 39
Ft, F, Fm’, Fm’-F, Y(II), Fv/Fm and Mean Value display... 396.2.1
Zero Offset and noise N(t) ................................................... 406.2.2
6.3Algae-window ........................................................................... 42
6.4Settings-window........................................................................ 44
6.5Slow Kinetics window .............................................................. 49
6.6Light Curve window.................................................................. 51
Edit Light Curves ................................................................. 536.6.1
Light Curve Fit-parameters .................................................. 566.6.2

CONTENTS
Comments on Light curves .................................................. 626.6.3
6.7Report Window ......................................................................... 64
6.8Reference-window deconvolution and Chlorophyll
determination calibration values ............................................... 68
How to generate Reference Spectra ..................................... 726.8.1
6.9Fluo Spec -window.................................................................... 74
6.10Fast Kinetik - window.......................................................... 75
O-I1Fit window .............................................................. 776.10.1
Parameters and Output of Fo-I1Rise Analysis ............... 816.10.2
6.11VIEW - MODE .................................................................... 82
6.12Main Menu Bar .................................................................... 85
Options: ETR Parameter ................................................. 876.12.1
Al current/PAR list ......................................................... 886.12.2
6.13Script files ............................................................................ 90
Data Management ........................................................... 916.13.1
Editing Tools................................................................... 926.13.2
List of Script File Commands ......................................... 936.13.3
7Technical Specifications........................................................... 101
PHYTO-PAM-II Compact Version ................................... 1017.1.1
System Control and Data Acquisition................................ 1027.1.2
Battery Charger MINI-PAM/L .......................................... 1037.1.3
Spherical Micro Quantum Sensor US-SQS/WB................ 1037.1.4
Transport Box PHYTO-T .................................................. 1047.1.5
Accessory Stirring Device WATER-S............................... 1047.1.6
8Rechargeable battery ............................................................... 105
9References and further reading .............................................. 106
10Index .......................................................................................... 112
11Warranty.................................................................................. 115

MAY 2002 CONTENTS
11.1Conditions .......................................................................... 115
11.2Instructions......................................................................... 116

CHAPTER 1MAY 2002 SAFETY INSTRUCTIONS
1
1Safety instructions
1.1 General safety instructions
1. Read the safety instructions and the operating instructions
first.
2. Pay attention to all the safety warnings.
3. Keep the device away from water or high moisture areas.
4. Keep the device away from dust, sand and dirt.
5. Always ensure there is sufficient ventilation.
6. Do not put the device anywhere near sources of heat.
7. Connect the device only to the power source indicated in the
operating instructions or on the device.
8. Clean the device only according to the manufacturer’s
recommendations.
9. If the device is not in use, remove the mains plug from the
socket.
10. Ensure that no liquids or other foreign bodies can find their
way inside the device.
11. The device should only be repaired by qualified personnel.

CHAPTER 1 SAFETY INSTRUCTIONS
2
1.2 Special safety instructions
1. PHYTO-PAM-II Multiple Excitation Wavelength Phyto-
plankton and Photosynthesis Analyzer is a highly sensitive
research instrument which should be used only for research
purposes, as specified in this manual. Please follow the
instructions of this manual in order to avoid potential harm to
the user and damage to the instrument.
2. PHYTO-PAM-II employs high intensity LED-array light
sources which may cause damage to the eye. Avoid looking
directly into these light sources during continuous illumination
or saturation pulses.
3. Do not cover the ventilation grille rear side of the instrument.

CHAPTER 2 PHYTO-PAM-II
3
2PHYTO-PAM-II
PHYTO-PAM-II represents the progress of established WALZ
fluorometers dedicated to aquatic research like XE-PAM, WATER-
PAM, previous PHYTO-PAM and MULTI-COLOR-PAM. Choice
of several measuring light wavelengths at variable measuring light
settings (intensity and frequency) offered by XE- and 4-wavelengths
PHYTO-PAM are combined with the high time resolution of
MULTI-COLOR-PAM. Thus enabling classical PAM analysis,
deconvolution of phytoplankton and analysis of fast induction
kinetics.
The PHYTO-PAM-II incorporates a multi-color Chip-On-Board
(COB) LED Array featuring 5 measuring light colors and 6 colors for
actinic light illumination. The 440 nm, 480 nm, 540 nm, 590 nm,
625 nm measuring light facilitates online differentiation of 4
different pigment types therefor deconvolution of green algae,
cyanobacteria, diatoms/dinoflagellates and phycoerythrin containing
organisms like cryptophytes. As in the first generation PHYTO-PAM
deconvolution bases on fluorescence excitation reference spectra.
But for the first time these spectra are not instrument specific but
universal. Due to spectral calibration of PHYTO-PAM-II instruments
reference spectra can be shared between users and instruments.
The PHYTO-PAM-II user surface is based on the proven PhytoWin-
software featuring classical PAM analysis like the estimation of the
effective photochemical quantum yield of PS II and the
determination of photochemical and non-photochemical quenching
parameters. For extended applications a special Fast Kinetics mode
of operation is added for measuring the wavelength-dependent O-I1
fluorescence rise kinetics upon onset of pulses of strong actinic light
information. Thus enabling evaluation of the functional absorption
cross-section of PS II, σPSII, determined by light color and the

CHAPTER 2 PHYTO-PAM-II
4
pigment composition of photosynthetic organisms (Klughammer and
Schreiber, 2015)
Two versions of the PHYTO-PAM-II Phytoplankton Analyzer are
available. A PHYTO-PAM-II Compact Version with integrated
emitter-detector unit and a Modular PHYTO-PAM-II Version
featuring different emitter-detector units connectable to a Power-and-
Control-Unit.
Both versions of the PHYTO-PAM-II are operated by PhytoWin-3
software.

CHAPTER 2 PHYTO-PAM-II
5
2.1 Compact Version
The PHYTO-PAM-II Compact Version is a lightweight and easily
portable instrument. The splash-proof cast aluminium housing
incorporates the control unit as well as all essentials of the emitter-
detector unit: the optical unit, the measuring and actinic LED-array,
the photomultiplier-detector and the cuvette retainer.
Fig. 1: PHYTO-PAM Compact Version
Front side:
Power switch and green power indicator lamp
USB socket to connect external PC for instrument control
Charge socket, to connect Battery Charger MINI-PAM/L
(100 to 240V AC)
Fuse plug, containing 3.15 AT main fuse of internal power
circuit

CHAPTER 2 PHYTO-PAM-II
6
Light Sensor socket, to connect the Spherical Micro
Quantum Sensor (US-SQS/WB) for PAR list calibration.
Top:
Measuring Head with optical port for inserting sample
cuvette
PVC centering ring with o-ring sealing against the inner
wall of the Measuring Head, serving as a guide for the
cuvette and as an adapter for mounting the optional
Miniature Stirring Motor Water-S and the optional Spherical
Micro Quantum Sensor US-SQS/WB.
Cup-shaped perspex inset sealing against an inner o-ring of
the Measuring Head, thus protecting the opto-electronical
components from spilled water samples.
Agreen LED shows the status of the internal
Photomultiplier Detector. The photomultiplier
automatically is switched off at excessive light impact.
Quartz Cuvette with 15 mm outer and 13 mm inner
diameter; height 46 mm.
Darkening Hood covering the part of the Measuring Head
protruding from the housing.
Photomultiplier Detector The Photomultiplier supply
voltage is automatically switched off when it sees too much
light (red status LED on housing lights up).
COB LED-Array The light emitting diodes (LED) are
densely arranged on a 10 x 10 mm area. PHYTO-PAM-II
provides 5 differently coloured measuring lights (440 nm,
480 nm, 540 nm, 590 nm, 625 nm), and 5 actinic light

CHAPTER 2 PHYTO-PAM-II
7
sources (440 nm, 480 nm, 540 nm, 590 nm, 625 nm); the
latter are complemented by white (440-640 nm) and far red
(735 nm) LEDs (for measuring light and actinic light spectra
see Fig. 2). To compensate for varying brightness of the
different types of light emission diodes, more LEDs are used
when the intensity of the individual LED is lower.
Fig. 2: Typical normalized emission spectra of PHYTO-PAM-II light
sources. Spectra are not corrected for spectral response of the
spectrometer.

CHAPTER 2 PHYTO-PAM-II
8
Spherical Micro Quantum Sensor US-SQS/WB2.1.1
Fig. 3: Spherical Micro Quantum Sensor US-SQS/WB
The Spherical Micro Quantum Sensor US-SQS/WB is included in
the system package as calibration of the PAR-List is essential for
correct assessment of all PHYTO-PAM-II functions.
The US-SQS/WB device consists of a submersible spherical
quantum sensor, US-SQS/L, and a small amplifier unit which is
connected to the light sensor socket. The amplifier is tuned to yield
correct PAR readings within the automated PAR-List calibration (see
chapter 3.1) and needs to be set to 1x.
The 3.7 mm diffusing sphere of the US-SQS/WB sensor picks up
photosynthetically active radiation (PAR) with an angular response
error of less than ± 5% from -100° to 100° angle. Hence, the sensor
is ideally suited to measure light conditions in the suspension cuvette
where reflections and scattering results in randomization of light
direction. For stability of calibration, it is important to keep the

CHAPTER 2 PHYTO-PAM-II
9
diffuser clean. If needed the diffuser can be gently cleaned by using a
cotton tip applicator moistened with some ethanol.
The position of the light sensor is adjustable and will be determined
for PHYTO-PAM-II applications by a 15.3 mm spacer above the
sensor hood.
Stirring Device WATER-S (optional)2.1.2
Fig. 4: Stirring Device WATER-S
WATER-S stirring device can be particularly useful for
measurements of rapidly settling samples.
A special adapter ring is provided for mounting the optional Stirring
Device WATER-S to the PHYTO-PAM-II Compact version.
WATER-S runs on a long life 3 V Lithium battery (size CR 123A). It
features an on/off switch and a potentiometer knob for stirring rate
adjustment. In the bottom center, a stirring paddle is mounted on the

CHAPTER 2 PHYTO-PAM-II
10
motor axis (via split brass-tube adapter). The disposable paddle can
be removed by gentle pulling. The other way around, a replacement
paddle can be mounted by pushing its cylindrical end all the way into
the holder. For replacement of the battery the housing has to be
opened by pulling the white and grey halves apart. Separation of the
two halves is facilitated by forcing gently a thin flat body into the slit
(like finger nail or thin screw driver).
It should be noted that at high photomultiplier gain the paddle of the
WATER-S will cause some increase of noise. This is due to the fact
that some measuring light is reflected from the paddle towards the
photodetector, such that the background signal is approximately
doubled and the electronic noise is correspondingly increased.
Furthermore, there is an increase of sample noise caused by the
movement of cells or cell groups.
Battery Charger MINI-PAM/L2.1.3
The Battery Charger MINI-PAM/L is provided for recharging the
internal lead-acid battery (12V/2Ah) of PHYTO-PAM-II. It is
connected to the Charge-socket on the front panel of the Power-
and-Control-Unit. The charger, which operates at input voltages
between 100 and 240V AC, features overload protection. Battery
voltage is displayed on the Settings-window of PhytoWin Software.

CHAPTER 3 INSTALLATION
11
3PhytoWin Software installation and
Phyto-PAM-II setup
PhytoWin-3 and manual versions can be downloaded from Walz web
page http://www.walz.com/downloads/overview.html. The
PhytoWin-3 program needs be installed on the PC going to be used
in conjunction with the PHYTO-PAM-II. At the end of the guided
installation procedure a Phyto-PAM_3 folder is created on the PC
with Data-directories of three different types of Phyto-PAM
Measuring Heads (Phyto US, Phyto ED and Phyto Compact Unit).
Into these directories all measured data will be written. Next to these
directories a script folder, the Phyto.exe file and after definition of
the used measuring head, a Phyto.cfg is located within the Phyto-
PAM_3 folder.
After download double click the file Setup.exe
After start of Setup.exe the Install Wizard is called up.
This wizard guides through the installation, at the end of which the
PhytoPAM-3 folder will be installed on the PC.
Links to PhytoPAM-3 Folder and to
PhytoWin-3.exe are established on the
desktop.

CHAPTER 3 INSTALLATION
12
Connect PHYTO-PAM-II to computer via USB-cable and start
PhytoWin program by clicking the PhytoWin-3 icon on the desktop.
The Start-window is displayed, showing the number of the current
PhytoWin version.
Following query appears:
to search for Com-Port and enter Measure Mode
to view and analyse data acquired by the PHYTO-PAM-II
and start the program in the viewing mode.
to quit the program.

CHAPTER 3 INSTALLATION
13
In both software operation modes (Measure
Mode and Viewing Mode) the selection of
the applied measuring head is essential, as
each measuring head features individual
parameters (e.g. relating to photomultiplier
sensitivity) that are stored in separate Data
directories. These parameters are essential
for correct storage and analysis of the measured data. After definition
of the measuring head the actual program is started with the 5-
channels excitation window being displayed.
NOTE:
When a computer installs the PhytoWin-3 user software first time for
a selected PHYTO-PAM-II measuring head, the internal PAR list
needs to be measured before bringing into operation. Calibrations
of the PAR-Lists and a valid .par file in the measuring head folder is
essential for correct assessment of PHYTO-PAM-II functions.
The default.par file is only for startup and not suitable for functional
operation.
The features of the PhytoWin-3 user software described in the
following sections apply to all measuring heads. Exceptions are
indicated.
3.1 Measure PAR Lists
To bring the PHYTO-PAM-II to operation first time the LED array
light parameters (PAR lists) need to be calibrated. The light list
measurements include calibration of the light sensor offset and

CHAPTER 3 INSTALLATION
14
readout of the complete LED array done in an automated calibration
procedure.
Switch on PHYTO-PAM-II and start PhytoWin-3
software. Please plug in the Spherical Micro Quantum
Sensor US-SQS/WB to the PHYTO-PAM-II light sensor
connector, the light sensor will be recognized and the
Measure PAR Lists button in the Settings Window
enabled.
At first use the light sensor might show an offset. This
offset will be eliminated during light sensor calibration
within the Measure Par Lists routine.
Fill distilled water into the quartz glass cuvette and
insert it in the PHYTO-PAM-II measuring head.
Insert the light sensor. The position of the light sensor is
critical for correct detection of PAR values. Therefor the
light sensor/darkening hood geometry is fixed by a 15.3
mm spacer and should not be altered.
Open the measure PAR routine by clicking the Measure
PAR Lists button (Settings window) and start Light
calibration process.