FMS 2 Overview
The FMS 2+ chlorophyll fluorimeter consists of a control unit housing all of the electronics, optics and light sources necessary to derive most common chlorophyll fluorescence parameters. These are optically linked to the sample by a statistically randomised fibre-optic cable that is positioned in the FMS/PTL PAR/temperature leafclip. The fibre-optic cable is also suitable for insertion into a range of sample containers such as oxygen electrode chambers, older gas-analysis chambers, petri dishes and microtitre plates.
The system may be operated in several different modes: serial connection to a Windows® PC enables real-time instrument control and data presentation. Captured data is simultaneously presented as a real-time chart recorder emulation and parameters-only format for easy identification of key experimental events. This PC mode of operation is suitable for development of complex protocols which may be programmed into the instrument using the simple drag-and-drop editor to generate user-defined scripts. These scripts automate the execution of experiments in the field, allowing complex protocols involving many control events to be operated with the same ease as single control event measurement such as . Once programmed, the FMS 2+ can be used as a stand-alone chlorophyll fluorimeter in either laboratory or field situations, running from internal batteries with all measurement data and calculated parameters saved to integral protected memory. The unit can store up to six experimental protocols, any one of which may be accessed and executed using the built-in menu system. When data collection is complete the results can be downloaded to the Windows® software for full analysis.
All of the light sources required for modulated measurement of common chlorophyll fluorescence parameters are self-contained within the instrument.
594 nm amber modulating beam with 4-step frequency control. (Optional 470 nm blue LED)
Dual-purpose ultra-bright LED light source providing actinic light (0 µmol m-2 s-1 – >4,000 µmol m-2 s-1 in 50 steps) and saturating pulse (0 µmol m-2 s-1 – >13,000 µmol m-2 s-1 at the sample surface in 100 steps)
735 nm far-red LED source for preferential PSI excitation allowing accurate determination of the
The PAR/temperature leafclip is available to facilitate measurements made under ambient light conditions using the FMS 2+ chlorophyll fluorimeter. Chlorophyll fluorescence measurements can be made quite satisfactorily without the leafclip but a value of PAR from the light sensor on the leafclip is essential for the estimation of electron transport rate () by the FMS software. Other chlorophyll fluorescence parameters are unaffected if the system is operated without the leafclip.
The FMS/PTL leafclip consists of a sprung upper section which gently grips the sample in a gentle clamping action. A grooved neck mounted at 60° to the plane of the sample accommodates the fibre optic cable which is slid into position.
Marked graduations on the neck can be aligned with graduations on the fibre-optic cable termination to reference its position for future work, a retaining screw locks it into position throughout the experiment. The rest of the fibre-optic cable may be looped over the leafclip and hooked to the rear of the clip to help support its weight. A fully cosine corrected PAR sensor and 0°C – 90°C thermocouple are also fitted to the FMS/PTL.
An electrical connection to the leafclip socket on the front panel of the FMS 2+ chlorophyll fluirometer enables the use of the remote trigger switch to activate/abort measurements in local mode and connect the leafclip thermocouple and light sensor to the control unit. The leafclip may be held in the hand if multiple samples are being studied or mounted on a standard tripod mount via a thread in the lower clip section for fixed-position work.
The PAR sensor has been designed for both recordings of ambient light intensities during fluorescence analysis and measurement of FMS actinic and saturating light sources during instrument setup.
Field-Swappable Battery System
The FMS 2+ chlorophyll fluorimeter is powered by a 2.0 Ahr lead-acid battery, capable of >8 hours of continuous measurement in the field. Batteries can be easily exchanged in the field as they become discharged.
2 batteries are supplied with the FMS 2+ which, when both fully charged, easily provide at least 2 full days measurement in the field. The FMS 2+ carrying bag has space in the bottom to carry the spare battery.
FMS/DLC Dark-Adaptation Clips
FMS 2+ is supplied with a set of 10 dark-adaptation leafclips (catalogue number FMS-DLC). These should be used when dark-adaptation of the sample (or measurement in the absence of light), is required before or during the experiment. The leafclips can be attached to samples independently prior to measurements to facilitate adequate dark-adaptation time for the sample being measured without hindering throughput.
Dark-adaptation leafclips can be used for a wide variety of different samples such as broad-leaf species (including Arabidopsis), grasses and needle species.
Dark-adaptation leafclips are constructed from 3D printed nylon. To dark-adapt the sample, a stainless steel shutter plate in the upper section of the clip is slid forward, covering the sample, and effectively preventing ambient light from reaching the leaf. During measurements, the sampling end of the fibre-optic cable is inserted into the fibre receptor on the upper section of the leafclip and securely fastened using the thumb-screw. Before taking measurements, the shutter is retracted to the open position, exposing the dark-adapted sample to the fibre-optic light source.
The sample rests on a foam pad whilst in the clip in order to minimise damage to the structure of the sample.
During dark adaptation, all the reaction centres are fully oxidised and available for photochemistry and any chlorophyll fluorescence yield is quenched. This process takes a variable amount of time and depends upon plant species, light history prior to the dark transition and whether or not the plant is stressed. Typically, 15 – 20 minutes may be required to dark adapt effectively. In order to reduce waiting time before measurement, a number of leaves may be dark adapted simultaneously using several leafclips.
Modfluor32 & Parview32 Software
PC control from Modfluor32 Windows® software allows real-time trace plotting as a chart-recorder emulation with calculated parameters written to a text parameters window. Instrument features and parameter measurement routines are selected from a toolbar with drop-down menus to control file handling and instrument configuration.
Complex experimental protocols may be automated to reduce repetitive work by developing scripts using an iconised script editor which allows a sequence of control functions and measurements to be developed into a protocol. Once created, scripts may be executed directly from the Modfluor32 program and data viewed while the instrument automatically completes a user-defined experiment. A maximum of six scripts can be downloaded to the instrument’s internal memory for operation without a computer.
The connection of an optional external battery to FMS 1+ (FMS 2+ is supplied with batteries) enables portable operation with data stored to instrument memory for subsequent upload and full graphical presentation on the computer.
A further application is also included with the FMS 1 and FMS 2+ chlorophyll fluorimeters. Parview32 is a stand-alone utility designed to allow easy upload and transfer of multiple parameter files to a spreadsheet type program.
FMS Parameters Measured
Dark adapted parameters measured
Dark adaptation inhibits all light-dependent reactions. The resulting absence of photochemistry for a sufficient length of time allows complete re-oxidation of PSII electron acceptor molecules, opening PSII reaction centers and thus maximising the probability that absorbed light can be used for photochemistry. Commonly measured parameters from tissue in this state are used to calculate the maximum quantum efficiency of PSII and are usually used to reference measurements made on light adapted samples.
– The fluorescence origin () is defined as the chlorophyll fluorescence yield following dark adaptation when all of the PSII reaction centers and electron acceptor molecules are fully oxidised and hence available for photochemistry. As a result, is often measured at the beginning of an experiment when only the modulating beam is illuminated.
– The maximum fluorescence yield () is attained when the dark adapted sample is exposed to an intense saturating pulse of light from the chlorophyll fluorometer. This temporarily reduces all PSII electron acceptors preventing PSII photochemistry. The temporary absence of competition from photochemistry for absorbed energy ensures maximum chlorophyll fluorescence emission from the sample.
– The difference between the and chlorophyll fluorescence yield relates the maximum capacity for photochemical energy quenching by the sample and is defined as variable fluorescence ().
– Calculation of the rate constants for competing energy dissipation pathways in tissue under dark-adapted () and light-saturated () conditions have shown that the ratio of variable to maximal chlorophyll fluorescence () is directly proportional to the quantum efficiency of PSII photochemistry (Butler 1977, 19781). Close correlation with other measures of quantum efficiency of photochemistry in a wide range of species (Björkman and Demmig 19872) has resulted in widespread use of as a screening parameter for stress response.
Light adapted parameters
In the case of light adapted tissue, a proportion of PSII electron acceptors are reduced, closing some PSII reaction centers. Hence the probability that absorbed energy is used for photochemistry is not maximal as competing non-photochemical processes are operating. The measurement of the light adapted ratio of variable to maximal chlorophyll fluorescence ratio permits the estimation of PS II quantum efficiency () using the model of Genty et al. 19893.
, and – Several measurements of fluorescence yield from the sample in different defined states are required to estimate . Initially, the fluorescence yield of the sample under the ambient light regime is required. Such measurements are often made after a sample has adapted to a particular light regime or environment and is operating at steady state. Consequently, the measurement is often referred to as the steady-state fluorescence yield or .
A fully saturating pulse from the chlorophyll fluorimeter is then required to close all of the PSII reaction centers; the temporary inhibition of PSII photochemistry ensures that the maximal fluorescence yield () is achieved. If a previous dark adapted measurement of has been made the extent of photochemical and non-photochemical quenching processes can be determined from the equations of Schreiber et al 19864.
and – Adaptation to high irradiance can involve significant changes in the confirmation of the photosynthetic apparatus which result in non-photochemical energy dissipation in the PSII antennae, before energy reaches the reaction centers (Horton et al. 19915, Horton and Ruban 19946). Failure to account for this effect can lead to inaccuracies in calculation of the relative contributions of photochemical and non-photochemical energy dissipation. This problem can be overcome by transiently shading the sample and using a far-red light source to preferentially excite PSI relative to PSII (electrons are drawn through the electron transport chain effectively opening PSII reaction centers and allowing measurement of a light adapted , usually given the notation .
Other parameters measured
– Takes a reading of the current incident photosynthetically active radiation value in µmol m-2 s-1 from the on-board PAR sensor of the FMS/PTL if connected to the chlorophyll fluorometer. Please note that the FMS/PTL leafclip is supplied as standard with FMS 2 but must be purchased as an option for FMS 1.
– Takes a reading of the current temperature in °C from the on-board thermocouple of the FMS/PTL if connected to the chlorophyll fluorometer. Please note that the FMS/PTL leafclip is supplied as standard with FMS 2 but must be purchased as an option for FMS 1.
– A measure of the photochemical quenching coefficient calculated as:
– A measure of the non-photochemical quenching coefficient calculated as:
– An alternative definition of non-photochemical quenching calculated as:
– A measure of the electron transport rate calculated as:
Measurement of requires the FMS/PTL leafclip which is supplied as standard with FMS 2+ but must be purchased as an option for FMS 1+.
1 Butler, W. L., (1977).
Chlorophyll fluorescence: a probe for electron transfer and energy transfer.
In Encyclopaedia of Plant Physiology, ed. A. Trebst, M. Avron, 5, 149-167. Berlin: Springer-Verlag.
Butler, W. L., (1978).
Energy distribution in the photochemical apparatus of photosynthesis.
Annual Review of Plant Physiology, 29, 345-378.
2 Björkman, O. and B. Demmig (1987).
Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77K among vascular plants of diverse origins.
Planta, 170, 489-504.
3 Genty, B., Briantais, J-M. and N.R. Baker (1989).
The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence.
Biochimica et Biophysica Acta, 990, 87-92.
4 Schreiber, U., Schliwa, W. and U. Bilger (1986).
Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorimeter.
Photosynthesis Research, 10, 51-62.
5 Horton, P., Ruban, A.V., Rees, D., Pascal, A. A., Noctor, G. and A. J. Young (1991).
Control of the light-harvesting function of chloroplast membranes by aggregation of the LHCII chlorophyll-protein complexes.
FEBS Lett., 292,1-4.
6 Horton, P. and A. Ruban (1994).
The role of light-harvesting complex II in energy quenching. In Photoinhibition of photosynthesis from molecular mechanisms to the field
ed. N. R. Baker and J. R. Bowyer pp. 111-128. Oxford: BIOS, Scientific Publishers Ltd.
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