Operation Manual for the FIRe Fluorometer System · Operation Manual for the FIRe Fluorometer System Document No. SAT-DN-00265 Revision G, 12 December 2012

Operation Manual for the FIRe Fluorometer System

Document No. SAT-DN-00265 Revision G, 12 December 2012

Revision: G SAT-DN-00265 Copyright © 2010 Satlantic Page: ii

Operation Manual For: FIRe Fluorometer System

Document Number: SAT-DN-00265

Satlantic LP Richmond Terminal, Pier 9 3481 North Marginal Road

Halifax, Nova Scotia B3K-5X8

Tel (902) 492-4780 Fax (902) 492-4781

Copyright © 2010, Satlantic

This document contains information proprietary to Satlantic or to a third party to which Satlantic may have legal obligation to protect such information from unauthorized disclosure, use or duplication. Any disclosure, use or duplication of this document, in whole or in part, or of any of the information contained herein for any purpose other than the specific purpose for which it was disclosed is expressly prohibited, except as Satlantic may otherwise agree to in writing.

SYSTEM

FIRe Fluorometer System

SECTION TABLE OF CONTENTS

Revision: G SAT-DN-00265 Copyright © 2010 Satlantic Page: iii

1. OVERVIEW .......................................................................................................................... 1-1 1.1. BACKGROUND............................................................................................................... 1-1 1.2. FEATURES.................................................................................................................... 1-2 1.3. PRINCIPLES OF OPERATION........................................................................................... 1-2 1.4. CALIBRATION ................................................................................................................ 1-3 1.5. OPERATIONAL SUMMARY............................................................................................... 1-3

2. START UP............................................................................................................................ 2-1 2.1. INSTALLING EMISSION FILTER ........................................................................................ 2-1 2.2. INITIAL CONNECTIONS ................................................................................................... 2-1 2.3. POWER REQUIREMENTS................................................................................................ 2-3

3. DATA ACQUISITION........................................................................................................... 3-1 3.1. INTRODUCTION ............................................................................................................. 3-1 3.2. FIREVIEW .................................................................................................................... 3-1 3.2.1. Starting a measurement ............................................................................................. 3-1 3.2.2. Saving a file ................................................................................................................ 3-2 3.2.3. Command Summary................................................................................................... 3-3 3.2.4. Adjustable Parameters ............................................................................................... 3-3 3.2.5. Reference Excitation Profile ....................................................................................... 3-5 3.2.6. Gain Set...................................................................................................................... 3-6 3.2.7. Delay Interval.............................................................................................................. 3-6 3.2.8. Number of Samples .................................................................................................... 3-6 3.2.9. Single Turnover Flash................................................................................................. 3-6 3.2.10. Single Turnover Relaxation Phase............................................................................. 3-7 3.2.11. Multiple Turnover Flash .............................................................................................. 3-7 3.2.12. Multiple Turnover Relaxation Phase........................................................................... 3-7 3.2.13. Configuration File........................................................................................................ 3-7 3.3. SAMPLING TIPS............................................................................................................. 3-7 3.3.1. Adjusting Signal Strength ........................................................................................... 3-7 3.3.2. Selecting the Gain ...................................................................................................... 3-7 3.3.3. Time Intervals ............................................................................................................. 3-9 3.4. USING THE FIBER OPTIC PROBE (FOP) ....................................................................... 3-11 3.4.1. Fiber Optic Probe Setup ........................................................................................... 3-11 3.4.2. Sampling with the Fiber Optic Probe........................................................................ 3-13 3.4.3. Instrument Care ........................................................................................................ 3-13 3.5. USING THE ACTINIC LIGHT SOURCE (ALS) ................................................................... 3-14 3.5.1. Manual PAR Acquisition ........................................................................................... 3-14 3.5.2. PAR Stepping Acquisition......................................................................................... 3-14

4. DATA PROCESSING........................................................................................................... 4-1 4.1. INTRODUCTION ............................................................................................................. 4-1 4.2. FIREPRO ..................................................................................................................... 4-1 4.3. AUTOMATIC BLANK CORRECTION................................................................................... 4-3 4.4. RESULTS FILE............................................................................................................... 4-3 4.4.1. Data File Description .................................................................................................. 4-3

5. CONTINUOUS MODE.......................................................................................................... 5-1 5.1. FIRECONT ................................................................................................................... 5-1

SYSTEM


SECTION TABLE OF CONTENTS

Revision: G SAT-DN-00265 Copyright © 2010 Satlantic Page: iv

5.2. COMMANDS .................................................................................................................. 5-1 5.3. FLOW THROUGH CUVETTE ............................................................................................ 5-3 5.3.1. Maintenance ............................................................................................................... 5-4

6. FILE MANAGER .................................................................................................................. 6-1 6.1. INTRODUCTION ............................................................................................................. 6-1 6.2. COMMANDS .................................................................................................................. 6-1

7. WARRANTY......................................................................................................................... 7-1 7.1. WARRANTY PERIOD ...................................................................................................... 7-1 7.2. RESTRICTIONS.............................................................................................................. 7-1 7.3. PROVISIONS ................................................................................................................. 7-1 7.4. RETURNS ..................................................................................................................... 7-1 7.5. LIABILITY ...................................................................................................................... 7-1

8. CONTACT INFORMATION ................................................................................................. 8-1 8.1. LOCATION..................................................................................................................... 8-1 8.2. BUSINESS HOURS......................................................................................................... 8-1

9. DECLARATION OF CONFORMITY.................................................................................... 9-1

10. APPENDIX A...................................................................................................................... 10-1 GAIN CALIBRATION .................................................................................................................. 10-1

11. APPENDIX B...................................................................................................................... 11-1 BLUE LED EXCITATION SPECTRUM........................................................................................... 11-1 GREEN LED EXCITATION SPECTRUM ........................................................................................ 11-2

12. APPENDIX C...................................................................................................................... 12-1 OUTPUT FILE ........................................................................................................................... 12-1 RESULTS FILE.......................................................................................................................... 12-2

SYSTEM


SECTION 1. OVERVIEW

Revision: G SAT-DN-00265 Copyright © 2010 Satlantic Page: 1-1

1. OVERVIEW

1.1. Background

The Satlantic FIRe (Fluorescence Induction and Relaxation) System is the latest advance in bio-optical technology to measure variable fluorescence in photosynthetic organisms. Developed in collaboration with Dr. Maxim Gorbunov and Dr. Paul Falkowski from Rutgers University, the system is based on 15 years experience in basic and applied research and development in photosynthesis and oceanography. The FIRe system can be used in the following applications:

• Basic and applied photosynthesis research

• Environmental monitoring of phytoplankton and benthic organisms (macrophytes, coral, sea grass, algal turfs)

• Water quality monitoring

• Assessment of primary productivity in aquatic ecosystems

• Assessment of environmental stresses

• Eco-physiology

Figure 1. FIRe Fluorometer System

SYSTEM


SECTION 1. OVERVIEW


1.2. Features

The Satlantic FIRe system comes equipped with the following components:

• Onboard computer, monitor, keyboard, 2 GB USB drive

• Removable filter package (680nm and 880nm)

• Two quartz cuvettes (1 cm id)

• FIReView logging software package

• FIRePro data analysis package

1.3. Principles of Operation

The FIRe technique relies on active stimulation and highly resolved detection of the induction and subsequent relaxation of chlorophyll fluorescence yields on micro- and millisecond time scales. To accommodate efficient excitation of diverse functional groups within phytoplankton communities including a variety of cyanobacteria, we have developed a multicolor excitation source. This source uses high luminosity blue (450 nm with 30 nm bandwidth) and green (530 nm with 30 nm bandwidth) light-emitting diodes (LEDs) to excite chlorophyll and bacteriochlorophyll fluorescence in vivo. A computer-controlled LED driver circuit generates pulses with the duration varied from 1 µs to 50 ms. Each LED generates up to 1 W/cm2 of peak optical power density in the sample chamber or at the leaf surface to ensure fast saturation of PSII within the single photosynthetic turnover (less than 50 µs).

The fluorescence signal is isolated by red (678 nm, with 20 nm bandwidth, for Chl-a fluorescence) or infra-red (880 nm with 50 nm bandwidth, for BChl-a fluorescence) interference filters and detected by a sensitive avalanche photodiode module. The fluorescence signal is amplified and digitized by a 12 bit analog-to-digital converter at 1 MHz sampling rate. To accommodate a wide range of Chl-a concentrations (from ~0.05 to 100 mg/m3) in natural phytoplankton and laboratory cultures, the gain of the detector unit can be adjusted over the range of three orders of magnitudes. An embedded low-power CPU board controls the excitation protocols and data acquisition and performs the real-time data analysis using a custom analysis toolbox.

An example of the FIRe measurement protocol consisting of four phases is shown in Figure 2. Phase 1 consists of a strong short pulse of 100 μs duration (called the Single Turnover Flash or STF) that is applied to cumulatively saturate PSII and measure the fluorescence induction from Fo to Fm(STF). During Phase 2, a weak modulated light is applied to record the relaxation kinetics of fluorescence yield on the time scale of 500 ms. The Multiple Turnover Flash begins with Phase 3 and a strong long pulse of 50ms duration applied to saturate the PSII and PQ pool. Finally, in Phase 4 a weak modulated light is applied to record the kinetics of the PQ pool re-oxidation on the timescale of 1s.

SYSTEM


SECTION 1. OVERVIEW


The analysis of fluorescence induction on microsecond time scales (Phase 1) provides the minimum (Fo) and maximum (Fm) fluorescence yields, the quantum efficiency of photochemistry in PSII (Fv/Fm), the functional absorption cross-section of PSII (σPSII), and the energy transfer between PSII units (‘connectivity factor’, p). The recorded relaxation kinetics of fluorescence yields reflects the rates of electron transport on the acceptor side of PSII and between PSII and PSI (Phase 2). The photosynthetic electron transport rates as a function of irradiance, together with coefficients of photochemical and non-photochemical quenching are measured using the new Actinic Light Source (ALS).

Figure 2. An example of the FIRe measurement protocol

1.4. Calibration

The FIRe system provides a relative measure of the functional absorption cross-section of PSII (σPSII). In order to determine the absolute functional absorption cross-section of PSII (σPSII), the user needs to apply the calibration coefficients supplied for the blue and green light sources. To obtain the calibration coefficients, please refer to the calibration sheet that was included with the FIRe shipment.

1.5. Operational Summary

To summarize, the FIRe measurement protocol consists of the following four phases:

A strong short pulse of 80 μs duration (called Single Turnover Flash, STF) is applied to cumulatively saturate PSII and measure the fluorescence induction from Fo to Fm(STF).

A weak modulated light is applied to record the relaxation kinetics of fluorescence yield on the time scale of 500 ms.

SYSTEM


SECTION 1. OVERVIEW


A strong long pulse of ca. 20 ms duration (called Multiple Turnover Flash, MTF) is applied to saturate PSII and the plastoquinone (PQ) pool.

A weak modulated light is applied to record the kinetics of the PQ pool re-oxidation the time scale of 1s.

Analysis of the Phase 1 provides Fo, Fm, Fv/Fm (STF), σPSII, p; Phase 2 – time constants for the electron transport on the acceptor side of PSII (i.e., re-oxidation of the Qa acceptor); Phase 3 – Fm (MTF) and Fv/Fm(MTF); Phase 4 – the time constant for the electron transport between PSII and PSI (re-oxidation of the PQ pool).

With the optional ALS (Section 3.4) users can also determine the analogous light adapted fluorescence measurements (Fo', Fs, Fm') and quenching parameters (qN, qP, and NPQ).

SYSTEM


SECTION 2. START UP


2. START UP

2.1. Installing Emission Filter

The FIRe Fluorometer System is equipped with a removable filter holder which houses two pre-installed emission filters (Fig 3). The two interference filters have central wavelengths of 678 nm (for Chl-a measurements) and 880 nm (for bacterial Chl measurements).

NOTE: To avoid damage to the detector by the intense light, the emission filter (either 678 nm or 880 nm) must be installed into the FIRe System prior to turning on the power.

For transportation, the filter must be removed from the instrument and packed separately to avoid damage.

Figure 3. Installing the removable emission filter in the filter holder

2.2. Initial Connections

Connect the power cord, monitor, and keyboard to the connectors on the back panel of the instrument as shown in Figure 4.

SYSTEM


SECTION 2. START UP


Figure 4. Rear panel connections on FIRe system

The main ON/OFF switch is on the front panel. The red LED indicator will go on and the PC DOS will be booted. The FIRe System is now ready for use. Turn ON the BLUE LED switch on the front panel for the blue LEDs to be used as an excitation source. Alternatively, Turn ON the GREEN LED switch on the front panel for the green LEDs to be used. You may have both switches turned on if you are interested in using both light sources at the same time.

Figure 5. Front panel on FIRe system

SYSTEM


SECTION 2. START UP


2.3. Power Requirements

The FIRe System can be powered with any AC voltage ranging from 85 to 250V and can be plugged in directly (without voltage converter) to an electrical outlet anywhere in the world. Depending on the country and the type of electrical outlet, an outlet adapter may be needed.

SYSTEM


SECTION 3. DATA ACQUISITION


3. DATA ACQUISITION

3.1. Introduction

The FIRe software consists of three different programs: FIReView, FIRePro, and FIReCont, currently at version 1.3.0.

If you wish to use an external USB drive for data transfer or software updates, you must plug it into the USB port on the front panel before turning on power so that the USB driver is properly initialized.

To get started, turn on the main power switch on the lower left-hand side of the front panel and press the <Enter> key when prompted to display the following menu:

• 1. FIReView • 2. FIRePRo • 3. FIReCont • 4. File manager • 5. Quit

3.2. FIReView

FIReView is the data acquisition program used to collect fluorescence profiles of discrete samples. It enables the user to manipulate adjustable parameters and control the data acquisition process.

3.2.1. Starting a measurement

To start acquiring data, use the arrow keys (←,→,↑,↓) to go to the 2nd line of the left menu ‘Log File:’ and enter the name of the destination file (i.e., the file where the fluorescence profile is to be saved, e.g., SAMPLE1.000). To enter the file name, press [Space Bar], type the name with extension (e.g., SAMPLE1.000), and press [Enter]. The file name must not exceed 8 letters/numbers (DOS requirement) and should have the extension .000. When a fluorescence profile is saved, the extension will increase automatically by one (.001, .002, and so on). Therefore, when logging a series of consecutive measurements, the user is not required to type a new file name for each individual measurement. Explanation of the adjustable fields can be found in Table 1.

To start the measurement, press the s key on the keyboard. The program will start the data acquisition until the preset number of acquisition ‘Number Samples:’ is reached. The data acquisition may be stopped by pressing s at any time.

SYSTEM




Figure 6. Typical fluorescence profile with proper gain setting.

If necessary, adjust the GAIN, and restart data acquisition (press r to reset the acquired signal and then s to start a new data acquisition). For a discussion on proper gain adjustment please refer to section 3.3.2.

3.2.2. Saving a file

To save the acquired fluorescence profile, press w (i.e., write). The data files (SAMPLE1.000, etc.) will automatically be saved in the same directory from which FIReView is run (i.e. C\:Fire). When the profile is saved, the extension of the destination file will automatically increase by one. The program is now ready to conduct the next measurement. To start a new data acquisition, press r and then s.

NOTE: If you do not press ‘w’ after completing a measurement it will be lost when a new measurement is performed.

When the measurements are completed, press q to quit the program.

SYSTEM




3.2.3. Command Summary

In summary, the following action keys can be used to control measurements:

a – ALS on/off toggle actinic light source on/off

r – Reset to clear the display

p – Save Profiles when this option is toggled on it saves the raw fluorescence traces for each measurement into a file

c – Ref Correction toggle the use of the reference excitation profile during the collection of the fluorescence profile

x – Collect Ref Exc toggle the collection of the reference excitation profile. If turning on reference excitation collection, the use of the reference excitation to normalize the profile is toggled off.

s – Start/Stop start/stop the measurement

w – Write Data save (write) the acquired profile

l – Load File load an existing data file for viewing

q – Quit quit the program

Note: The action keys are Case Sensitive. If [Caps Lock] is ON, the program does not respond.

3.2.4. Adjustable Parameters

This section describes the parameters used in the measurement protocols and provides guidelines for how and when they may be changed. The FIReView logging program starts with a default excitation and measurement protocol that has been selected and optimized to suit the most common applications. The default settings are stored in the Setup.txt file in the C:\Fire directory. The user can edit the default protocol by changing the Setup.txt file using the File Manager program. Alternatively, the user can adjust the parameters onscreen by highlighting the desired variable (hit <spacebar>) and entering new values (followed by <Enter>). The following table lists the adjustable parameters.

SYSTEM




Table 1. FIReView Adjustable Parameters

Parameter Default Description

Exc Profile Exc0.txt This is the reference excitation profile that FIReView uses to normalize the variable fluorescence profile. The default file is suitable for use with GAIN values of 0 to 2000. For the highest GAIN setting, the Exc2400.txt file is recommended.

Log File a.000 The name of the file which data is written to. It consists of a root name and an extension based on 000. The extension number increments automatically with each sample written to file.

LED Color Blue The user enters the led color used. This value is saved in the data file header.

Gain 0 Relative gain of the detector. May vary from 0 to 2400. To determine the absolute gain value, see appendix A for the gain calibration

Sample Delay 1000 Time interval between consecutive acquisitions (msec). Can be increased to allow for physiological “recovery” between flash sequences.

Number Samples

10 Number of acquisitions in a given sequence.

STF 80 Length of the single turnover flash (pulse) in μsec.

STRP 40 Number of pulses in the relaxation sequence

STRI 60 The initial interval between relaxation pulses in μsec

MTF 20 Length of the multi-turnover flash phase in ms.

MTRP 40 The number of pulses in the relaxation sequence

MTRI 100 The initial interval between relaxation pulses.

Comments Enter user defined comments. These comments are included in the header of the data file.

PAR 0 Sets the maximum PAR value for the actinic light source. Valid range is 0-2000 μMol/m2/s.

SYSTEM




Num PAR Steps

0 Sets the number of steps to use when logging data in PAR stepping mode. The PAR value entered is divided by the number of PAR steps and then the PAR level is incremented by this amount starting at 0 and ending at the maximum PAR value.

PAR Off Int 0 Sets the time delay (sec) between PAR levels when using PAR stepping. This option turns off the ALS between steps for the duration entered. Can be left at 0 if user does not want this option.

PAR On Int 0 Sets the time delay (sec) in which the actinic light source is on prior to data acquisition. Flash sequence will be delayed by this timed setting allowing for adaptation to the new PAR level. Can be left at 0 if user does not want this option.

Actinic Light Off Indicates whether the actinic light source is on or off.

3.2.5. Reference Excitation Profile

The FIReView program is used to store a reference excitation profile (measured at Satlantic with a fluorescent dye) that is used to normalize the sampled variable fluorescent profiles. The default file (Exc0.txt) is loaded automatically and is suitable for use with gain settings between 0 and 2000. For the maximum gain setting, it is recommended that the Exc2400.txt file be used.

Fluorescence yield is defined as the ratio of the fluorescence intensity to the excitation light intensity. Calculation of the temporal evolution in the fluorescence yields, therefore, requires the accurate knowledge of the excitation profile (called “reference”). The optical output of the Light Emitting Diodes used in the FIRe System is stable and reproducible, that eliminates the need for the reference profile to be recorded at each measurement. The default FIRe reference excitation profile is recorded and saved in the FIRe System memory (file EXC0.txt). However, because the system characteristics may slightly change over time, the customer may want to re-acquire the reference from time to time.

To update the reference excitation profile, run the FIReView program by selecting option “1” from the main menu for FIReView. Use a fluorescent dye solution (e.g. uranine or extracted chlorophyll) at a concentration that produces a strong signal at the desired GAIN setting. To achieve a high signal-to-noise ratio, a large number of acquisitions should be used (at least 20). Ensure that collect reference excitation is turned on (type “x” to toggle the collection of a reference excitation profile). Type “s” to start the program and save the new file with a unique name (e.g. EXCBLUE.txt or EXCGREEN.txt). To use the new reference excitation profile, select the “Exc Profile” field and update to the new file.

SYSTEM




Since the reference may potentially differ between blue and green LED sources, acquisition and use of the two individual reference profiles (for blue and green, respectively) is recommended.

3.2.6. Gain Set

To accommodate a wide range of Chl-a concentrations in natural phytoplankton and laboratory cultures, the electronic gain of the detector unit is adjustable over the range of two orders of magnitude (see Appendix A). The GAIN (in relative units) can be varied in the range from 0 to 2400 and is selected from the main program menu. We suggest the GAIN to be changed with an increment of 200 (i.e., 0, 200, 400,…, 2000, 2400). In this case, the measurements conducted at different GAIN values can easily be compared by using the conversion table in Appendix A. The actual electronic Gain Value is a nonlinear function of GAIN.

3.2.7. Delay Interval

The delay interval ‘Sample Delay:’ enables the user to adjust the amount of time between subsequent samples. This will be a trade off between enabling the Fo value to recover after the saturating pulse of the MTF and sampling frequency. Obviously, the longer the delay, the better fluorescence recovery, but the lower the sampling rate.

3.2.8. Number of Samples

The number of acquisitions or ‘Number Samples’ allows the user to vary the number of repeated profiles taken for a single fluorescence profile. If the fluorescence signal is low (e.g. oligotrophic waters) relative to the noise, a large number of acquisitions will increase the signal to noise ratio and provide better results. In more dense populations (e.g. near shore or laboratory cultures), the number of samples can be greatly reduced.

If the “save profiles” setting is toggled on, these repeated profiles are, in addition to being used in creating the fluorescence profile, themselves kept. When the user saves the profile by typing the “w” command, the repeated profiles are also stored in the ouput file.

3.2.9. Single Turnover Flash

The single turnover flash (STF) duration is set to a default value of 80 μs. This duration can be adjusted up or down depending on the shape of the STF induction curve. If the fluorescence profile does not reach saturation plateau by the end of the STF (small σPSII), the time may be lengthened. If the saturation peak is reached very early in the STF (large σPSII), the STF may be shortened to ensure that multiple turnovers are not induced.

SYSTEM




3.2.10. Single Turnover Relaxation Phase

This option enables the user to adjust the number of the weak modulated pulses in the relaxation phase of the STF to ensure that the fluorescence profile reaches a low plateau before the beginning of the MTF. The software default is 40 with 60 µsec separation between the pulses.

3.2.11. Multiple Turnover Flash

The multiple turnover flash duration is adjustable to ensure that the photosynthetic apparatus of the sample cells are undergoing several turnovers and the Fm(MTF) parameter reaches a plateau. The default value is 20 ms.

3.2.12. Multiple Turnover Relaxation Phase

This option enables the user to adjust the number of the weak modulated pulses in the relaxation phase of the MTF to ensure that the fluorescence profile reaches a low plateau before the beginning of the next sequence. The software default is 40 with 100 µsec separation between the pulses.

3.2.13. Configuration File

The default settings are stored in the ASCII file SETUP.txt and may be changed prior to the startup of the FIReView program (e.g. using the command EDIT SETUP.txt from DOS prompt).

3.3. Sampling Tips

3.3.1. Adjusting Signal Strength

To increase the signal-to-noise ratio, multiple samples may be needed. The number of samples is therefore the number of complete sequences of pulses. Number of samples may vary from 1 (single short) up to several hundred. While a single short acquisition (Number Samples = 1) may be sufficient to measure variable fluorescence in dense laboratory cultures, number of samples of up to 50-100 may be required for low chlorophyll samples in oligotrophic regions of the ocean.

3.3.2. Selecting the Gain

When the fluorescence signals are very low and the GAIN is maximum, the Y-scale of the screen is automatically magnified by a factor of 1 to 10, depending on the signal magnitude. This procedure is similar to the “Digital Zoom” option in modern digital cameras (incorporating both optical zoom and digital zoom) and helps the user to visualize the low signals in real-time.

SYSTEM




While working with dense laboratory cultures of algae, we suggest that the user start measurements at the minimum GAIN (= 0) and increase the GAIN until the optimal signal is achieved. When the algal sample is too dense and the fluorescence signal is out of range even at the minimum GAIN, the sample has to be diluted or a neutral density filter installed in combination with the emission red filter. Note that at very high sample chlorophyll densities, the excitation light may be significantly attenuated (self-shading effect) even on the short path of the cuvette (~1 cm) and the results will be compromised.

With continuous measurements using FIReCont, the GAIN is automatically adjusted by the program in increments of 400.

Figure 7. Fluorescence profile with gain set too low

SYSTEM




Figure 8. Fluorescence profile with the gain set too high

FAQ: Why isn’t the GAIN automatically adjusted for discrete sample analysis? - For specific experiments, customers may want to select the GAIN manually and, e.g., conduct a series of measurements at a fixed GAIN.

When the GAIN is too low the measurements are still valid. However, the signal-to-noise ratio may be reduced and the precision of the readings may be compromised (Figure 7). For the maximum precision, the fluorescence profile should reach the upper part of the screen (between the middle of the screen and the red line on the top). When the GAIN is too high and the signal is out of screen the readings are no longer valid (Figure 8).

3.3.3. Time Intervals

The time interval between acquisitions is required for the fluorescence yield to relax down to its minimum level between acquisitions. The default excitation protocol consists of a strong short (~80 μs) pulse of light, called Single Turnover Flash (STF), and a strong long (~20 ms) pulse, called Multiple Turnover Flash (MTF). Each STF and MTF is followed by weak modulated light to record the relaxation kinetics of fluorescence yield

SYSTEM




(STRP and MTRP). The parameters listed in Table 1 define the excitation protocol and may be changed from the main menu.

To employ only the Single Turnover protocol, MTF and MTRP must be set to 0.

SYSTEM




3.4. Using the Fiber Optic Probe (FOP)

Figure 9. Fiber Optic Probe for the FIRe System

The fiber-optic probe is an external FIRe accessory that can be used to measure variable fluorescence for situations in which it is not practical to place individual samples into the FIRe itself. Examples of such situations would be sampling solids, such as leaf surfaces or culture racks where the user has many samples lined up. The Fiber optic probe uses a randomized fiber bundle to ensure that the excitation light field is uniform and that the maximum amount of variable fluorescence is measured.

3.4.1. Fiber Optic Probe Setup

Visually inspect the optical surfaces to ensure that they are clean and free from scratches.

Connect the male serial connector on the fiber optic probe to the port labeled “External Probe” on the rear faceplate of the bench top FIRe.

Place the variable fluorescence return path branch of the fiber optic probe into the cuvette holder (Fig 10).

SYSTEM




Figure 10. Insert the fluorescence return end of the probe into the cuvette holder of the bench-top FIRe instrument.

The probe guide should be adjusted so that the variable fluorescence return path branch of the fiber optic probe sits firmly in the cuvette seat. The probe guide can be adjusted by loosening its two thumbscrews and sliding the guide up or down (Fig 11). Once adjusted, tighten the thumbscrews to keep the probe guide in place.

Figure 11. Adjust the probe guide so that the tip fits securely in the cuvette seat.

The fiber optic probe is now ready for use.

SYSTEM




3.4.2. Sampling with the Fiber Optic Probe

Start the bench-top FIRe and run FIReView. Configure FIReView as you would for normal operation.

Place the tip of the sample branch of the fiber-optic probe against the surface of the sample (e.g. leaf, water sample). The tip guard on the end of the probe provides a 1 mm buffer between the probe lens and the sample. This is the calibrated distance and is only important if absolute measurements are required. The fiber-optic probe calibration is only valid when the sample branch is held at this distance.

Press ‘s’ to start the data acquisition process.

FIReView works exactly the same regardless of whether samples are measured internally with the cuvette or with the fiber optic probe. However, since the flash excitation is controlled by the external probe port in the rear panel and delivered by the probes own LED, the ‘Blue’ and ‘Green’ LED switches do not have an effect on the FOP pulse sequence. The FOP box contains its own LED and thus does not rely on the bench top FIRe for the excitation energy.

3.4.3. Instrument Care

Due to the delicate internal optical components, the Fiber Optic Probe requires careful handling. Misuse of this device can result in deterioration of performance over time. In general please keep the following points in mind when using the Fiber Optic Probe:

Bending the fiber bundle into a tight radius may cause some of the internal fibers to break, thus reducing the sensitivity of the instrument.

Impacts or physical shocks to the fiber jacket may damage internal fibers

Impacts to the electronic housing may damage the internal electronics and/or optical components.

SYSTEM




3.5. Using the Actinic Light Source (ALS)

It is possible to attach and use an actinic light source (ALS) in order to achieve constant levels of photo-synthetically active radiation (PAR) in the sample cuvette. The ALS (Figure 12) is connected via the serial port located on the back of the computer case.

Figure 12. Actinic Light Source for the FIRe System

The actinic light source has two modes for acquiring data, manual PAR acquisition and stepping PAR acquisition.

3.5.1. Manual PAR Acquisition

This mode is the simpler of the two. The user selects a PAR setting by selecting the ‘PAR (µmol/m2/s)’ option and inputting the desired value. Allowable PAR values range from 0 - 2000 µmol/m2/s. Once the PAR value is set, simply select ‘a’ on the keyboard to toggle the actinic light source on, followed by toggling ‘s’ to start data acquisition. All the related PAR parameter settings are included in the header of the logged data file. Once the data has been acquired select ‘w’ to write the data output file.

3.5.2. PAR Stepping Acquisition

With PAR stepping acquisition the user sets up a range of PAR values through which the data acquisition steps. Each step represents a specific PAR intensity that increases incrementally with each acquisition event. During each step the data is collected through a series of iterations just like normal data logging. Each PAR step with its associated iterations is called a data acquisition event and is logged to a separate data file. Data logging with PAR stepping is automatic and does not require using the ‘w - Write Data’ key.

In order to enable PAR stepping both the ‘PAR (µmol/m2/s):’ and ‘Num PAR Steps:’ options must have valid values.

SYSTEM




Setup for PAR stepping is as follows:

Set a value for the maximum attained PAR value in the ‘PAR (µmol/m2/s)’ option. Allowable PAR values range from 0 – 2000 µmol/m2/s. PAR stepping will start at a PAR value of 0 µmol/m2/s and then gradually increase the PAR value incrementally until reaching the maximum set PAR value.

Enter the number of PAR increments desired by entering a value into the ‘Num PAR Steps’ option. This will determine how many data acquisition events will occur and the increasing intensity of the PAR values with each acquisition event. For example, setting a PAR value of 2000 µmol/m2/s and then setting the number of steps to NSteps: 3 will result in four data acquisition events with PAR levels of 0, 1000, and 2000 µmol/m2/s. A separate data log file will be automatically created for each data acquisition event.

Occasionally, it is desirable to have the ALS turn off for a certain time period between data acquisition events. Entering a value in seconds into the ‘PAR Off Int (sec)’ option will set this delay. When a data acquisition event is complete the ALS will turn off and no data will be logged during the defined interval. Note that during the first acquisition event the ALS is turned off, therefore the second acquisition event will proceed without the delay. All subsequent acquisition events that have the ALS turned on will observe the delay period between events.

It is sometimes desirable to have the ALS turned on for a certain period of time immediately prior to a data acquisition event. Entering a value in seconds into the ‘PAR On Int (sec)’ option will set this delay. The ALS will turn on for this length of time and when completed data acquisition will begin with the ALS remaining on.

The user can still control the number of iterations within a data acquisition event by entering a value into the ‘Number Samples’ option. Note that in PAR stepping this value does not represent the total amount of iterations. Instead it represents the amount of iterations for each data acquisition event.

The user can still set the delay value in milliseconds between each acquisition by entering a value into the ‘Sample Delay (msec)’ option.

Once all the desired parameters are set, select ‘s’ on the keyboard to begin PAR stepping data acquisition. A separate data log file is written for each data acquisition event. The labeling of the files increases incrementally the same as in normal data logging.

During the acquisition process the ‘Num PAR Steps’ option will count down the number of data acquisition events until reaching 0. The ‘PAR (µmol/m2/s)’ option will display the current PAR value for the acquisition event. The “PAR (µmol/m2/s):’ value will increase with each acquisition event until reaching the maximum value for the last event.

The user can stop the data acquisition events by pressing ‘s’ on the keyboard.

SYSTEM


SECTION 4. DATA PROCESSING


4. DATA PROCESSING

4.1. Introduction

The FIRe system comes with a data analysis program called FIRePro. The logged data files can be analyzed on the FIRe systems internal computer or they can be downloaded to a separate PC. To speed the data processing, the later is recommended.

4.2. FIRePro

FIRePro enables the user to resolve the following parameters from the fluorescence profile obtained with FIReView:

Fo, Fm Minimum and maximum yields of chlorophyll-a fluorescence measured in a dark-adapted state

Fv Variable fluorescence (= Fm- Fo )

Fv / Fm Maximum quantum yield of photochemistry in PSII, measured in a dark-adapted state

σPSII Functional absorption cross section of PSII

p ‘Connectivity factor’, defining the excitation energy transfer between individual photosynthetic units

Fo', F', Fm' Minimum, steady-state, and maximum yields of chlorophyll-a fluorescence measured under actinic light

ΔF' / Fm' Quantum yield of photochemistry in PSII, measured under actinic light (= (Fm'-F')/Fm' )

Fv'/ Fm' Quantum efficiency of photochemistry in open reaction centers of PSII, measured in a light-adapted state (= (Fm'- Fo')/ Fm' )

qp Coefficients of photochemical quenching (= (Fm'- Fs)/( Fm'- Fo') )

qN Coefficients of non-photochemical quenching (= (Fm - Fm')/( Fm - Fo) )

τQa Time constant for the electron transport on the acceptor side of PSII (the time of Qa re-oxidation)

τPQ Time constant for the electron transport between PSII and PSI (the time of the PQ pool re-oxidation)

SYSTEM




Note: To run the program on a separate PC, copy the program (FIRePro.exe), together with the file EGAVGA.bgi (a Turbo Pascal graphic driver). The program does NOT run without this graphic driver. The program FIRePro must be stored in the same directory as the data files.

To begin processing data, start the data analysis program select option #2 from the main menu. From the FIRePro main window, enter the name of the first data file to be processed (e.g. ST1.000) next to the Log: field. To enter the file name, press [Space Bar] and when finished typing the name, press [Enter]. This will update the Res: field with the new name of the output file (e.g. ST1.res). If you want the output file to have a different base name, enter the new name next to the Res: field.

Figure 13. Fitting profile in FIRePro

To begin processing, type i to import the data file. The program will start processing all the files in the series, that is files that have the same root name with an incrementing suffix such as.000, .001, .002 etc. Note that the number of iterations used during the processing is configurable by using the arrow keys to move to the Iterations field, pressing [Space Bar], typing the required number of iterations, and finally pressing the [Enter] key.

SYSTEM




STRP and MTRP processing may be disabled by using the s and m commands respectively. Note that disabling of the STRP processing also disables the MTRP processing.

Note: If there is a break in the extension number sequence (e.g. A.003 is missing), the program breaks after the last consecutive file (i.e., A.002). Then you have to restart the program and enter the name of the next available file (i.e. A.004).

4.3. Automatic Blank Correction

An automatic blank correction function is provided in FIRePro. This function subtracts a blank fluorescence yield from a sample fluorescence yield prior to curve fitting and parameter calculation. To use this function, enter a blank sample filename into the field labeled “Blank File” and toggle the “Blank” option to “ON”.

A blank sample is recorded in FIReView using the same procedure for any sample. Simply give it a descriptive name and enter it into the FIRePro program. Please note that the blank sample should be recorded with the same setup parameters as the samples to which it will be linked. For example, if a series of samples are recorded with a STF of 80 μs, a MTF of 600 samples, at a GAIN of 800, the blank should also be recorded with these values as well.

A blank measurement is most accurate when using a filtered sample of the seawater (or media) in which you are working. This will capture any dissolved fluorescing entities (e.g. CDOM) that may be present in the solution. When working in the field, we recommend checking the blank on a daily basis. If access to filtered seawater or culture media is restricted, ultra-pure de-ionized water may be used as a substitute.

4.4. Results File

FIRePro automatically saves the calculated parameters (Fo, Fm, Fv/Fm,…) to the specified results file. The result file can then be opened into Excel spreadsheet for further analysis.

4.4.1. Data File Description

Table 2. Description of data fields for the *.RES file output by FIRePro.

Parameter Description

Date Date when the sample was measured

Time Time (in seconds) of the day the measurement was taken

Time (s) = Hour * 3600+Min * 60.

SYSTEM




PAR The setting used for the actinic light source (µmol/m2/s)

Fo Minimum fluorescence yield (relative units) 1

Fm Maximum fluorescence yield calculated from the Induction (saturation) portion of the Single Turnover Flash (STF) profile (relative units)

FmDEC Maximum fluorescence yield calculated from the Relaxation portion of the Single Turnover Flash profile (relative units)2

Fv Variable fluorescence (= Fm – Fo)

FvDEC = FmDEC – Fo

Fv/Fm = (Fm-Fo)/Fm

FvD/FmD = (FmDEC-Fo)/FmDEC

Sigma The functional absorption cross section of PSII (σPSII)

p “Connectivity factor” (dimensionless), the parameter defining the energy transfer between PSII units.

Alp1, Alp2, Alp3 Relative amplitudes of the individual components in the relaxation kinetics of the fluorescence yield following the STF flash.

Tau1, Tau2, Tau3 Time constants (microseconds) of the individual components in the relaxation kinetics of the fluorescence yield following the STF flash

Tauav1 Average time for the two fastest (Tau1 and Tau2) components

Tauav2 Average time for the three (Tau1, Tau2, and Tau3) components

Fm(MTF) Maximum fluorescence yield calculated from the Induction portion of the Multiple Turnover Flash (MTF) profile (relative units)

GAIN Gain of the APD detector

MTF: Alp1, Alp2 Relative amplitudes of the individual components in the relaxation kinetics of the fluorescence yield following the

SYSTEM




MTF flash.

MTF: Tau1, Tau2 Time constants (microseconds) of the individual components in the relaxation kinetics of the fluorescence yield following the MTF flash.

Notes to the results file:

When the measurements are conducted under background actinic light, the minimum fluorescence yield should be denoted as Fs (instead of Fo), and the maximum yield as Fm’ (instead of Fm). This is according to the accepted nomenclature of variable fluorescence parameters (van Kooten and Snel, 1990).

FmDEC is often higher than Fm (a minute step between the induction and relaxation portion of the FIRe protocol) because the strong saturating light during the induction part produces triplet quenchers that reduce (‘quench’) the recorded fluorescence yield. Shortly (within tens of microseconds) after the termination of the induction flash, the quenchers relax and the resulting fluorescence yield increases (from Fm to FmDEC).

The MTF portion of the results file is not filled in when MTRP processing has been disabled. See section 4.2.

SYSTEM


SECTION 5. CONTINUOUS MODE


5. CONTINUOUS MODE

5.1. FIReCont

FIReView and FIRePro are used for logging and processing discrete samples. The FIRe system can also be run continuously using the program FIReCont. This program is identical to FIReView in its operating principles and differs only in the execution. Once the program is set to sample, it samples continuously until it is directed to stop. This program is most often used with the flow through cuvette.

Figure 14. FIReCont main screen

5.2. Commands

There are six command options on the right-hand side of the FIReCont main screen that are used to run the program. They are:

c – Cont a – Autogain s – Start/Stop

SYSTEM




f – Fit r – Reset q – Quit

For typical continuous mode operations, we recommend that both Cont and Autogain functions be left in the On setting.

c – Cont This toggle option controls the continuous sampling operation of the program. The default setting is on (indicated by a red background); to turn off, type “C”. When this option is turned off, the program may be used to measure discrete samples, similar to FIReView.

a – Autogain The Autogain toggle option controls the automatic gain switching function. The default setting is on (indicated by a red background); to turn off, type “a”. When the option is turned off the GAIN setting will remain fixed.

s – Start/Stop The “s” key is used to start and stop the program. Once the program is started, it will commence the flashing protocol and repeat at successively higher gain settings until a strong signal is achieved. Once it has an acceptable signal, it will acquire the set number of acquisitions (e.g. N-Acq = 5). Once the last acquisition is completed, the program then goes through the fitting process and saves the results in an output file before starting the next acquisition. Pressing the “s” key during this process will stop the program

f – Fit The fitting function is performed automatically when the “Cont” setting is selected on. If this setting is off and the user is taking discrete samples, the “f” command may be used to fit the acquired fluorescence profile. It can not be toggled if the “Cont” setting is on.

r – Reset The “Reset” command puts the “Cont” and “Autogain” setting in their default state and clears the screen of any plots that may be displayed.

q – Quit Typing “q” will quit the program and return to the main menu. This has no effect while the program is acquiring and processing data while in continuous mode.

SYSTEM




5.3. Flow Through Cuvette

The FIRe flow through cuvette was designed to enable researchers to sample from a continuous flow water source. It consists of a quartz cuvette mounted in a Delrin housing with stainless steel tubes for inflow and outflow of sample water. The long tube that reaches closest to the bottom of the cuvette should be used as the inflow to ensure maximum mixing and flushing of the sample. We recommend keeping the flow rate to a minimum to reduce generation of bubbles. A gravity feed line from a sample reservoir is sufficient to ensure efficient flushing. The outflow tube can then be connected to a return feed or to a drain.

The flow through cuvette is installed in the FIRe cuvette holder in the same manner as the discrete cuvette. Simply place the flow through cuvette in the sample port on top of the main FIRe unit.

Figure 15. Installing the flow through cuvette.

SYSTEM




5.3.1. Maintenance

For optimum performance, it is critical that the flow through cuvette is cleaned and dried after each use. Although the metal tubes are made from the highest quality stainless steel, corrosion can occur if they are left in contact with seawater. To clean the tubes, flush with ultra pure distilled water for 5 minutes and dry tubes with forced air.

To clean the inside of the cuvette, remove the 4 screws on the top of the Delrin holder and carefully clean with an alcohol solution.

Figure 16. Flow through cuvette installed in cuvette holder with inflow and outflow tubes connected

SYSTEM


SECTION 6. FILE MANAGER


6. FILE MANAGER

6.1. Introduction

To view, copy or manipulate files, select option 5 from the main menu to enter the File Manager program. From this program the user may manipulate data files as well as setup files.

6.2. Commands

Table 3. Command summary for File Manager program

Command Description

Space Mark file

F1 Copy marked files

F2 Delete marked files

F3 Move marked files

F4 Rename marked files

ENTER View highlighted file

CTRL-N Sort by name

CTRL-S Sort by size

CTRL-E Sort by extension

Left & Right Arrow Change drives (e.g. between C:\ and USB D:\)

CTRL-Z Help menu

Esc Exit

Data can be off-loaded using the USB port (D:\). To activate the USB port, you must have a USB drive plugged in at the time of startup. If it is not present when the instrument is initially powered up, the necessary USB drivers will not be initialized.

SYSTEM


SECTION 7. WARRANTY


7. WARRANTY

7.1. Warranty Period

All Satlantic equipment is covered under a one-year parts and labor warranty from date of purchase.

7.2. Restrictions

Warranty does not apply to products that are deemed by Satlantic to be damaged by misuse, abuse, accident, or modifications by the customer. The warranty is considered void if any optical or mechanical housing is opened. In addition, the warranty is void if the warranty seal is removed, broken or otherwise damaged.

7.3. Provisions

During the one year from date of purchase warranty period, Satlantic will replace or repair, as deemed necessary, components that are defective, except as noted above, without charge to the customer. This warranty does not include shipping charges to and from Satlantic.

7.4. Returns

To return products to Satlantic, whether under warranty or not, contact the Satlantic Customer Support Department and request a Returned Material Authorization (RMA) number and provide shipping details. All claims under warranty must be made promptly after occurrence of circumstances giving rise thereto and must be received by Satlantic within the applicable warranty period. Such claims should state clearly the product serial number, date of purchase (and proof thereof) and a full description of the circumstances giving rise to the claim. All replacement parts and/or products covered under the warranty period become the property of Satlantic LP.

7.5. Liability IF SATLANTIC EQUIPMENT SHOULD BE DEFECTIVE OR FAIL TO BE IN GOOD WORKING ORDER THE CUSTOMER'S SOLE REMEDY SHALL BE REPAIR OR REPLACEMENT AS STATED ABOVE. IN NO EVENT WILL SATLANTIC LP BE LIABLE FOR ANY DAMAGES, INCLUDING LOSS OF PROFITS, LOSS OF SAVINGS OR OTHER INCIDENTAL OR CONSEQUENTIAL DAMAGES ARISING FROM THE USE OR INABILITY TO USE THE EQUIPMENT OR COMPONENTS THEREOF.

SYSTEM


SECTION 8. CONTACT INFORMATION


8. CONTACT INFORMATION If you have any problems, questions, suggestions or comments about the equipment or manuals, please contact us.

8.1. Location Satlantic LP Richmond Terminal, Pier 9 3481 North Marginal Road Halifax, Nova Scotia B3K 5X8 Canada PHONE: (902) 492-4780 FAX: (902) 492-4781. Email: [email protected] Web: http://www.satlantic.com

8.2. Business Hours

Satlantic is normally open for business between the hours of 9 AM and 5 PM Atlantic Time. Atlantic Time is one hour ahead of Eastern Time. Daylight saving time is in effect from 2:00 a.m. on the second Sunday in March through 2:00 a.m. on the first Sunday in November. Atlantic Standard Time (AST) is UTC-4. Atlantic Daylight Saving Time (ADT) is UTC-3.

Satlantic is not open for business during the following holidays: • New Year's Day January 1st • Good Friday The Friday before Easter Sunday • Victoria Day The first Monday before May 25th • Canada Day July 1st • Halifax Natal Day The first Monday in August • Labor Day The first Monday in September • Thanksgiving Day The second Monday in October • Remembrance Day November 11th • Christmas Day December 25th • Boxing Day December 26th

SYSTEM


SECTION 9. DECLARATION OF CONFORMITY


9. DECLARATION OF CONFORMITY

SYSTEM


SECTION 10. APPENDIX A


10. APPENDIX A

GAIN calibration

The GAIN setting in FIReView is in relative units. The GainValue is the GAIN in absolute terms. Please use the table below to calculate the appropriate GainValue for your measurements.

GAIN (relative)

GainValue (absolute)

0 3.05

200 3.6

400 5.5

600 8.85

800 13.5

1000 20.9

1200 32

1400 50

1600 78.3

1800 123

2000 195

2200 283

2400 330

Note: The Gain function may vary between instruments and is subject to change.

SYSTEM


SECTION 11. APPENDIX B


11. APPENDIX B

Blue LED Excitation Spectrum

Blue LED Output

0

200

400

600

800

1000

1200

400 410 420 430 440 450 460 470 480 490 500

wavelength (nm)

uW/c

m2/

s

The above plot demonstrates that the peak wavelength for the blue LED’s is approximately 450 nm. The above spectra were measured under a constant current of 100 mA and therefore, the intensity scale is not indicative of that seen during pulsed sampling mode. For the measured PAR value under sampling mode, please refer to the calibration coefficients published in the Calibration section.

SYSTEM


SECTION 11. APPENDIX B


Green LED Excitation Spectrum

Green LED Output

0

50

100

150

200

250

300

350

500 510 520 530 540 550 560 570 580 590 600

wavelength (nm)

uW/c

m2/

s

The above plot demonstrates that the peak wavelength for the green LED’s is approximately 540 nm. The above spectra were measured under a constant current of 500 mA and therefore, the intensity scale is not indicative of that seen during pulsed sampling mode. For the measured PAR value under sampling mode, please refer to the calibration coefficients published in the Calibration section.

SYSTEM


SECTION 12. APPENDIX C


12. APPENDIX C

Output File

The data file logged by FIReView can be viewed/edited with any text editor or spreadsheet program and contains four columns of data. The first column is an incremental sample counter. The second column is elapsed time measured in μs. The third column is the reference excitation profile (see section 3.2.5 Reference Excitation Profile) and the fourth column is the relative fluorescence profile normalized to the reference excitation, or the fluorescence yield. During processing, the FIRePRo program loads the fluorescence yield data and enters the curve fitting routine to calculate the output parameters.

PC/104 FIRe Fluorometer 1.3.0 8/10/2006 49651 LED Color: blue Gain: 800 Sample Delay (msec): 1000 Number Samples: 5 STF (usec): 80 STRP (#samples): 40 STRI (usec): 60 MTF (ms): 20 MTF (#samples): 250 MTRP (#samples): 40 MTRI (usec): 100 PQLen: 4 Actinic Light Source Power: Off PAR (uMol/m^2/s): 0.00 Comment: Time EX Fl-Yield DATA-------------------------- 0 0 0 0.0 1 1 485 389.7 2 2 903 485.7 3 3 963 507.6 4 4 972 518.9 5 5 975 524.5 6 6 980 527.1 7 7 981 536.0 8 8 983 542.4 9 9 986 545.4 10 10 989 552.5 11 11 987 556.0 12 12 989 573.7 ↓ ↓ ↓

SYSTEM


SECTION 12. APPENDIX C


↓ ↓ ↓

Results File

Below is an example of an excerpt of processed data from the .RES file. This data can be viewed using MSExcel opening the file with “fixed width” columns.

File DATE TIME PAR Fo Fm FmDec Fv Test.000 12/1/2005 11828 0 653.1 1130.9 1239.5 477.7Test.001 12/1/2005 11964 0 705.3 1221.4 1338.7 515.9Test.002 12/1/2005 12052 0 670.1 1160.3 1271.7 490.1Test.003 12/1/2005 12907 0 723.7 1253.1 1373.5 529.3Test.004 12/1/2005 13052 0 687.5 1190.5 1304.8 502.9Test.005 12/1/2005 13180 0 742.5 1285.7 1409.2 543.1

FvDEC Fv/Fm FvD/FmD Sigma p Alp1 Tau1 Alp2 586.4 0.42 0.47 320 0.06 0.68 654 0.21633.3 0.46 0.51 346 0.06 0.73 706 0.23601.6 0.43 0.49 328 0.06 0.70 671 0.22649.8 0.47 0.52 355 0.07 0.75 725 0.23617.3 0.44 0.50 337 0.06 0.72 688 0.22666.7 0.48 0.54 364 0.07 0.77 744 0.24

Each successive sample saved in FIReView (i.e. 001, 002, 003…) shows up in the results file as an additional row. Therefore, all the data from a particular series is saved in the same results file.

Documents

Operation Manual for the FIRe Fluorometer System · Operation Manual for the FIRe Fluorometer System Document No. SAT-DN-00265 Revision G, 12 December 2012