opti-sciences OS1p User manual

OS1p User’s Guide
(Preliminary Advanced Version with RLCs for Algae and Higher Plants)
The New Standard in Portable
Chlorophyll Fluorometers
OS1p040111Advanced

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CHAPTER 1 •INTRODUCTION....................................................................................................................... 6
OVERVIEW...........................................................................................................................................................6
WELCOME ! .........................................................................................................................................................7
LIST OF EQUIPMENT.............................................................................................................................................7
GETTING STARTED...............................................................................................................................................7
CHLOROPHYLL FLUORESCENCE...........................................................................................................................8
DEFINITIONS:.....................................................................................................................................................11
QUANTUM PHOTOSYNTHETIC YIELD OF PSII –AN IN DEPTH DISCUSSION OF ITS VALUE AND LIMITATIONS. .......16
QUENCHING MEASUREMENTS,AN OVERVIEW ...................................................................................................21
Quenching equations:..................................................................................................................................24
Definitions - lake model parameters............................................................................................................25
Definitions - puddle model parameters........................................................................................................26
Understanding the quenching mode trace ...................................................................................................28
More helpful hints for setting test variable in quenching protocols. ...........................................................33
DARK ADAPTATION –HOW LONG IS LONG ENOUGH? .......................................................................................35
RELATIVE ELECTRON TRANSPORT RATE ...........................................................................................................37
BIBLIOGRAPHY ..................................................................................................................................................40
CHAPTER 2 •THE OS1P.................................................................................................................................. 52
INTRODUCTION ..................................................................................................................................................52
KEY FEATURES ..................................................................................................................................................53
PHYSICAL FEATURES PANEL PHOTOGRAPHS ...................................................................................................54
HARDWARE OVERVIEW AND MEASUREMENT PRINCIPLES .................................................................................56
LIGHT SOURCES .................................................................................................................................................57
Modulated light source ................................................................................................................................57
Saturation pulse light source .......................................................................................................................57
The LED actinic light source .......................................................................................................................58
The far-red light source ..............................................................................................................................58
THE FIBER OPTIC LIGHT GUIDE .........................................................................................................................58
ELECTRONICS ....................................................................................................................................................59
HOUSING AND CARRYING CASE.........................................................................................................................59
CHAPTER 3 •OPERATING THE OS1P......................................................................................................... 60
INTRODUCTION ..................................................................................................................................................60
INTRODUCTION TO RUNNING TESTS....................................................................................................................60
Set up menu..................................................................................................................................................61
Adjust screen touch sensitivity .....................................................................................................................62
Touch Panel Calibrate.................................................................................................................................62
Test menu .....................................................................................................................................................63
FV/FM PROTOCOL (OR FV/M ON THE SCREEN)..................................................................................................64
Cookbook check list before making Fv/Fm measurements..........................................................................65
Running the Fv/Fm test................................................................................................................................67
Drill down view of Fv/Fm measuring screens .............................................................................................68
Changing Fv/Fm measuring parameters -...................................................................................................69
Setting the modulation light source intensity...............................................................................................69
New -automated modulation light set up .....................................................................................................69
Far red light.................................................................................................................................................70
Setting the saturation pulse intensity ...........................................................................................................70
Setting saturation pulse duration.................................................................................................................70
Entering a note with a measurement............................................................................................................72

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Loading and saving preset measuring routines ...........................................................................................73
Other Fv/Fm function buttons......................................................................................................................74
Measurement review ....................................................................................................................................75
Help screen ..................................................................................................................................................75
Basic definitions of parameters....................................................................................................................76
Error messages ............................................................................................................................................76
Y(II) PROTOCOL:QUANTUM PHOTOSYNTHETIC YIELD OF PSII Y(II) OR )F/FM’ ............................................77
Cookbook checklist before making Y(II) measurements. .............................................................................78
Running the Yield of PSII Y(II) Test.............................................................................................................81
Drill down menu for Y(II) ............................................................................................................................82
Loading and saving preset measuring routines ...........................................................................................83
Copy presets parameters into measuring file...............................................................................................84
Change Y(II) measuring parameters............................................................................................................85
Setting modulation light source intensity.....................................................................................................85
New -automated modulation light set up .....................................................................................................86
Setting the saturation pulse intensity ...........................................................................................................86
Setting saturation pulse duration.................................................................................................................86
Multi-flash vs. standard single flash saturation pulse .................................................................................89
Far red light.................................................................................................................................................91
Pre-actinic light ...........................................................................................................................................92
Entering a note with a measurement............................................................................................................94
Other Y(II) or yield function buttons............................................................................................................95
Measurement review ....................................................................................................................................96
Help screen ..................................................................................................................................................97
Basic definitions...........................................................................................................................................97
Relative Electron Transport Rate.................................................................................................................98
e capture and quantum eff – setting leaf absorption & PSII ratio...............................................................98
Error messages ............................................................................................................................................99
QUENCHING PROTOCOL –HENDRICKSON –KLUGHAMMER LAKE MODEL:.....................................................100
Cookbook checklist before making quenching measurements..................................................................101
Running the quenching test........................................................................................................................105
Screen drill down diagram for quenching .................................................................................................106
Saturation pulse duration...........................................................................................................................107
Setting saturation pulse duration...............................................................................................................107
Saturation pulse count ...............................................................................................................................109
Saturation pulse interval............................................................................................................................109
Fo’ or Fod mode........................................................................................................................................109
Saturation pulse intensity...........................................................................................................................110
Modulation light source intensity...............................................................................................................111
New -automated modulation light set up ...................................................................................................111
Far red intensity and duration...................................................................................................................112
Actinic intensity..........................................................................................................................................112
Using Default PAR.....................................................................................................................................112
e capture and quantum eff – setting leaf absorption & PSII ratio.............................................................113
Log Set Up..................................................................................................................................................113
Enter a note................................................................................................................................................114
Measurement review ..................................................................................................................................115
Test pre-set files. Saving and loading test parameters...............................................................................116
Error messages common to the test modes ................................................................................................116
RAPID LIGHT CURVES (RCL)...........................................................................................................................118
How they work:..........................................................................................................................................119
Curve fitting software.................................................................................................................................120
Cardinal points description........................................................................................................................120
Saturation Pulse Duration .........................................................................................................................121
What are the limitations of RLC?...............................................................................................................122

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Cookbook checklist before making Rapid Light Curve measurements......................................................124
Running the RLC Test ................................................................................................................................126
Drill down menu for RLC...........................................................................................................................127
Saving measuring parameters....................................................................................................................128
Loading and saving preset measuring routines .........................................................................................129
Copy presets parameters into a new measuring file ..................................................................................129
Change RLC measuring parameters..........................................................................................................130
Setting modulation light source intensity...................................................................................................130
New -automated modulation light set up ...................................................................................................131
Setting the saturation pulse intensity .........................................................................................................131
Setting saturation pulse duration...............................................................................................................132
Far red light...............................................................................................................................................133
When the PAR Clip is used.........................................................................................................................134
When a PAR Clip is not used “Default PAR”............................................................................................135
Multi-flash vs. standard single flash saturation pulse ...............................................................................136
Relative Electron Transport Rate...............................................................................................................139
Entering a note with a measurement..........................................................................................................141
Other RLC function buttons.......................................................................................................................141
Measurement review ..................................................................................................................................142
Help screen ................................................................................................................................................143
Basic definitions.........................................................................................................................................143
Error messages ..........................................................................................................................................143
CHAPTER 4 •OS5P DATA MANAGEMENT..............................................................................................144
USB DATA TRANSFER ......................................................................................................................................144
File transfer by USB cable.........................................................................................................................145
Ejection process.........................................................................................................................................149
FILE TRANSFER BY SD CARD AND DATA MANAGEMENT UTILITIES.................................................................150
DATA VIEWER .................................................................................................................................................155
SOFTWARE UPDATES .......................................................................................................................................157
CHAPTER 5  DIAGNOSTICS................................................................................................................... 158
APPENDIX A •MAINTENANCE .................................................................................................................. 159
CLEANING........................................................................................................................................................160
MISCELLANEOUS MAINTENANCE.....................................................................................................................160
Battery........................................................................................................................................................160
Circuit breaker...........................................................................................................................................160
Light sources..............................................................................................................................................160
Trouble shooting power problems .............................................................................................................161
Trouble shooting tables..............................................................................................................................162
APPENDIX B•PAR CLIP ...............................................................................................................................165
PAR CLIP ........................................................................................................................................................165
Connection.................................................................................................................................................165
Technical specs ..........................................................................................................................................165
Using the PAR Clip....................................................................................................................................166
What is the value of a PAR clip in photosynthesis measurement?.............................................................167
APPENDIX C •TECHNICAL SPECIFICATIONS...................................................................................... 168

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APPENDIX D •DATA FORMATS................................................................................................................. 171
OVERVIEW.......................................................................................................................................................171
Data format information............................................................................................................................172
Fv/Fm data file format...............................................................................................................................172
Y(II) data file format ..................................................................................................................................173
Quenching data file format – Hendrickson – Klughammer equations.......................................................174
RCL Rapid Light Curve data format..........................................................................................................175

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Chapter 1 •Introduction
Overview
This chapter provides you with a list of the equipment that you should have received with your OS1p,
and Information about chlorophyll fluorescence with scientific references.
To select the best chlorophyll fluorescence measuring parameter for your application, consult
the Plant Stress Guide provided as a separate document.
The Plant Stress Guide is available on the CD provided with the OS1p or by visiting
www.optisci.com under Stress Testing, and Receive a Stress Guide with References.

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Welcome !
Congratulations on your purchase of the OS1p Modulated Fluorometer. Please check the
carton for any visible external damage. If you notice any damage, notify the freight carrier
immediately. Follow their procedures for reporting and filing a claim. The carton and all
packing materials should be retained for inspection by the carrier or insurer.
List of Equipment
Carefully unpack the carton. You should have received the following items:
•OS1p Fluorometer
•Universal Voltage battery charger
•Trifurcated fiber optic light guide with built-in trigger switch
•Ten dark-adaptation cuvettes
•Open body cuvette
•OS1p owner’s manual (this document)
•1 GB MMC/SD data card
•USB cable
•USB SD card reader
•Nylon field bag
If any item is missing, please contact your authorized Opti-Sciences agent.
Getting Started
Throughout this manual, you will be shown setup options and response messages. When an
example of a program screen is given, you may assume that this is shown, as it will appear on
the OS1p.
The user interface consists of a high-resolution color graphic touch screen LCD.
For editing parameters and making measurements, menu options are presented as icons or text
legends. Measuring tests and parameter adjustments are all menu driven. Certain keywords
are used to identify common functions. For example, the word or icon Exit will always step
you to the previous program screen, ultimately ending in the "Main Menu".
The OS1p has default settings that allow the unit to work for many applications, however,
changing the settings are very easily done.
Data is stored in on-board system memory. This is based on flash memory so no data will be
lost if the main battery is depleted. Stored data may be transferred to other systems through
use of the MMC/SD data card, or USB port. The data is output in comma delimited and
carriage return separated ASCII strings, easily importable to most spreadsheet programs such
as Excel and Mat Lab.

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Chlorophyll Fluorescence
A generalized description as it relates to chlorophyll fluorometry.
The following is a generalized description of the photosynthesis light reaction and the value
of chlorophyll fluorescence for investigation of plant health, plant function and plant stress.
Changes in photosystem II (PSII) fluorescence have been shown to be a sensitive test for most
types of plant stress, and reflect measurable changes of many plant functions including
photochemistry, photoprotective mechanisms, low light survival state transition mechanisms,
and heat dissipation photoinhibition mechanisms. PSII fluorescence measurements have been
found to correlate well with changes in CO2fixation under most conditions (for more detailed
correlation information refer to the section on “quantum photosynthetic yield of PSII”).
Reaction centers are of two types, Photosystem II (PSII), and Photosystem I (PSI). Both are
located in the thylakoid membrane of chloroplasts in higher plants. In bacteria, they are in a
membrane surrounding the cytoplasm or in more intricate constructs. All plants that produce
oxygen have both types of reaction centers.
While Photosystem I goes through a somewhat similar process to PSII, PSI fluorescence does
not vary with plant stress nor does it change as changes occur with various photosynthetic
mechanisms. Therefore PSII fluorescence is used for investigation into these areas.
Light energy utilized in photosynthesis by higher plants and algae cells is collected first by an
antenna pigment system and transferred to reaction centers where light quanta are converted
to chemical energy by chlorophylls in a protein environment. Electron transfer starts in the
reaction center when a chlorophyll molecule transfers an electron to a neighboring pigment
molecule. Pigments and protein involved in this primary electron transfer define the reaction
center. This initial electron transfer is also called charge separation.
Competing models of energy capture and transfer have existed. In the puddle model, each
reaction center possesses its own independent antenna system. In the lake model, reaction
centers share antenna. The “lake model” is considered more realistic for terrestrial plants.
PSII and PSI reaction centers also share antenna during a process called state transitions. This
process takes between ten and twenty minutes as a subset of PSII antenna detach from PSII
reaction centers and migrate to PSI reaction centers (Ruban, Johnson 2009). They can also
move back to PSII reaction centers. The process is governed by the oxidation-reduction state
of the plastoquinine pool, and it is thought to be a survival mechanism for plants to subsist in
low light conditions by balancing light levels between the two types of reaction centers
(Allen, Mullineau 2004). Light level changes and changing light quality can trigger
transitions.
During dark adaptation, higher plants and algae shift toward state 1 conditions and
cyanobacteria to state 2 conditions. (Papageorgiou G.C. Tismmilli-Michael M. Stamatakis K.
2007). State transitions should be considered when deciding on dark adaptation times and for
determination of steady state photosynthesis for quantum photosynthetic yield measurements,
and quenching measurements. State transitions affect measurements more at low light levels
than at high light levels (Lichtenthaler 1999).

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It is in PSII that oxygen evolution and the splitting of water occurs. In the presence of light
energy, an electron is pulled from a water-splitting complex and is used to reduce a PSII
reaction center.
Charge separation occurs as QA, the primary plastoquinone receptor, is reduced to QA-.
Electrons flow from there to other nearby plastoquinone molecules in the thylakoid membrane
by oxidation-reduction reactions. They act as energy transfer molecules in an electron
transport chain.
The next stop is a cytochrome b6f complex where a proton is supplied to the thylakoid lumen.
These protons and those supplied by the splitting of water are used by an ATP pump in the
thylakoid membrane, in the presence of ATP synthase, to create ATP from ADP. Eventually
the cytochrome b6f complex also supplies an electron to PSI.
PSI then goes through a somewhat similar process to PSII, and eventually produces NADPH
at the end of PSI. Both ATP and NADPH are used as energy sources to drive the dark
reaction, called the Calvin Benson Cycle of carbon fixation.
Factors such as light levels, light quality, water availability, nutrient availability, heat, cold,
herbicides, pesticides, pollution, disease, and genetic make up can all have an impact on CO2
assimilation, plant health and condition. When these factors are not provided at optimal levels
plant stress occurs, and most of these types of plant stress are also reflected in the
fluorescence signal from PSII (For more detailed information about the best test for specific
types of plant stress, consult the Plant Stress guide supplied on this disc or visit
www.optisci.com under stress testing).
In 1989, Bernard Genty found that there is a linear correlation between fluorescent quantum
photosynthetic yield measurements in C4plants and CO2assimilation. In 1990, Genty found a
curve linear correlation between fluorescent quantum photosynthetic yield and CO2
assimilation in C3plants, where photorespiration can also use significant products of electron
transport. Psydo-cyclic electron transport and other electron sinks may also be involved.
Different types of plant stress affect PSII differently, therefore one should consult the Plant
Stress Guide on this disc or contact Opti-Sciences at www.optisci.com to determine the best
measuring protocol or special assay before working with a specific type of plant stress.
Research referenced in the Plant Stress guide shows that while some types of plant stress
affect chlorophyll fluorescence of a plant in a dark adapted state (Fv/Fm), measuring some
types of plant stress, at a sensitive useful level, requires the light adapted Y(II) or )F/Fm’.
The Plant Stress guide is updates on a regular basis. For an updated version contact Opt-
Sciences Inc at www.optisci.com.
The most prominent antenna pigment that absorbs energy is usually chlorophyll a. Other
accessory pigments can also be involved, such as chlorophyll b., carotenoids, or phycobilins
in cyanobacteria, or bacteriochlorophyll in some bacteria. We are primarily concerned with
chlorophyll a fluorescence.
Light energy absorbed initially by the antenna and transferred to the reaction centers is
channeled to competitive, different plant processes that include photochemistry, photo-
protective heat dissipation, and other heat dissipation. Normally a healthy plant will channel

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about 3%-9% (Govindgee 2004) of the light energy absorbed by chlorophyll pigments as
chlorophyll fluorescence. Healthy plants in a healthy environment will use most light energy
for photochemistry. More stressful environments will channel additional energy to heat
dissipation and fluorescence.
This effect was first observed more than 100 years ago, when N.J.C. Müller (1874), using
colored glass filters, studied the phenomenon. He also noted that fluorescence changes that
occur in green leaves was related to photosynthetic assimilation. Lack of appropriate
technical equipment prevented a more detailed investigation.
The basic OS1p is equipped to make several different kinds of tests including: Dark-adapted
Fv/Fm, Light adapted Yield (Y) or )F/Fm’ or Y(II), Fo, Fm, Fms (or Fm’), Fs (or F’).It will
also measure fluorescence quenching, and rapid light curves.
PAR Clips are sold separately, and are required for ETR, PAR, and Leaf temperature.
PAR Clips are highly recommended for field measurement of Y(II) and ETR. Because Y(II)
values vary not only with plant stress, but also with light level and temperature, only samples
at very similar light levels and light histories should be compared. Sun leaves will respond
differently than shade leaves to different light levels.
The ability to use Rapid light curves will be provided in 2011 without additional charge for
those that buy the advanced version of the OS1p. The advanced version also allows the user
to select quenching protocols of interest when the unit is purchased. Other Protocols may also
be purchased during or after the initial purchase. Fod (or Fo’) are provided in the Kramer, and
puddle model quenching protocols. The default quenching protocol that is offered on the
advanced version of the OS1p is the Luke Hendrickson – Klughammer simplified lake model.
This provides the most versatile quenching solution because it includes NPQ that has been
resurrected from the puddle model. Other protocols are available for an additional charge.
One may replace the Hendrickson protocol with either the Kramer protocol or the puddle
model protocol at the time of purchase for no additional charge.
The quenching options include:
1. Dave Kramer’s lake model parameters (Kramer 2004) - Y(II), qL, Y(NPQ), and Y(NO)
2. Luke Hendrickson’s lake model parameters with Klughammer’s resurrection of NPQ from
the puddle model are included in this protocol - Y(II), Y(NPQ),Y(NO), and NPQ.
3. Puddle model quenching parameters qN, qP, and NPQ.
4. A relaxation protocol for subdividing NPQ into qE, qT, and qI. This protocol may be used
with NPQ in either the puddle model, or the Hendrickson lake model. The test runs at the
same time as the protocols mentioned above. To use this protocol either the Hendrickson or
puddle model protocols must also be purchased.