Field Portable Pulse Modulated Chlorophyll Fluorometer
- Pulse-modulated system
- Compact Field Design
- Temperature Compensated Electronics
- Programmable by Hansatech Scripting Language (HSL)
- Leaf-clip with integral PAR/temperature sensor
- Field Swappable Battery System
- Windows® data acquisition & data analysis software
The FMS 2 chlorophyll fluorometer 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 electrodes, 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 Fv/Fm. Once programmed, the FMS 2 can be used as a stand-alone chlorophyll fluorometer 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 halogen light source providing actinic light (0 – 3000 µmol m-2 s-1 in 50 steps) and saturating pulse (0 – 20,000 µmol m-2 s-1 in 100 steps)
- 735 nm far-red LED source for preferential PSI excitation allowing accurate determination of the Fo’ parameter
FMS/PTL PAR/Temperature Leafclip
The PAR / temperature leafclip is available to facilitate measurements made under ambient light conditions using the FMS 2 chlorophyll fluorometer. 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 – 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 fluorometer enables 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 recording of ambient light intensities during fluorescence analysis and measurement of FMS actinic and saturating light sources during instrument setup.
Dark Adaptation Leafclips
A leafclip system has been developed for situations where ambient light is to be excluded from the sample during measurement using the FMS chlorophyll fluorometer. This is suitable for experiments requiring dark-adapted measurements e.g. screening applications measuring Fv/Fm or situations which require adaptation of tissue to standardised doses of actinic light.
The system consists of small, lightweight leafclips and 2 different types of fibre optic cable adapter. The leafclip itself has a small shutter plate which should be closed over the leaf when the clip is attached so that light is excluded and dark adaptation takes place. The body of the clips are constructed from white plastic to minimise the effects of heat build-up on the leaf during the period when the clip is in place.
The locating ring section of the clip which interfaces with the fibre optic adapter is also constructed from white plastic.
The sample rests on a foam pad whilst in the clip in order to minimise damage to the structure of the sample. The shutter plate should be closed to exclude light from the sample during dark adaptation.
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.
The fibre optic cable is inserted into either one of the adapters which in turn, fits over the locating ring of the leafclip. The closed fibre optic adapter is suitable for applications where ambient light must be excluded whilst the open adapter is suitable for studies under ambient conditions.
FMS2 Field Swappable Battery System
The FMS 2 chlorophyll fluorometer is powered by a 2.0 Ahr lead-acid battery, capable of 1 hour maximum of continuous actinic illumination or of delivering approximately 800 saturating pulses.
Up to four batteries may be charged in the FMS 2 multi-charger overnight and easily exchanged in the field as they become discharged.
5 batteries are supplied with the FMS 2 which easily provide a full days measurement in the field. The FMS 2 carrying bag has space in the bottom to carry one spare battery.
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 with Hansatech Scripting Language (HSL). An iconised Script Editor 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.
Connection of an optional external battery 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 chlorophyll fluorometer. Parview32 is a stand-alone utility designed to allow easy upload and transfer of multiple parameter files to a spreadsheet type program.
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 centres 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 (Fo) is defined as the chlorophyll fluorescence yield following dark adaptation when all of the PSII reaction centres and electron acceptor molecules are fully oxidised and hence available for photochemistry. As a result Fo is often measured at the beginning of an experiment when only the modulating beam is illuminated.
The maximum fluorescence yield (Fm) 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 Fo and Fm chlorophyll fluorescence yield relates the maximum capacity for photochemical energy quenching by the sample and is defined as variable fluorescence (Fv).
Calculation of the rate constants for competing energy dissipation pathways in tissue under dark-adapted (Fo) and light-saturated (Fm) conditions have shown that the ratio of variable to maximal chlorophyll fluorescence (Fv/Fm) is directly proportional to the quantum efficiency of PSII photochemistry (Butler 1977, 1978*). Close correlation with other measures of quantum efficiency of photochemistry in a wide range of species (Björkman and Demmig 1987**) has resulted in widespread use of Fv/Fm 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 centres. 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 (PSII) using the model of Genty et al. 1989***.
Fs, Fm’ and ΦPSII
Several measurements of fluorescence yield from the sample in different defined states are required to estimate ΦPSII. 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 Fs.
A fully saturating pulse from the chlorophyll fluorometer is then required to close all of the PSII reaction centres; the temporary inhibition of PSII photochemistry ensures that the maximal fluorescence yield (Fm’) is achieved. If a previous dark adapted measurement of Fv/Fm has been made the extent of photochemical and non-photochemical quenching processes can be determined from the equations of Schreiber et al 1986$.
Fo’ and ΦPSIIR
Adaptation to high irradiance can involve significant changes in the conformation of the photosynthetic apparatus which result in non-photochemical energy dissipation in the PSII antennae, before energy reaches the reaction centres (Horton et al. 1991$$, Horton and Ruban 1994)$$$. 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 centres and allowing measurement of a light adapted Fo, usually given the notation Fo’.
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.
Takes a reading of the current temperature in °C from the on-board thermocouple of the FMS/PTL if connected to the chlorophyll fluorometer.
A measure of the photochemical quenching co-efficient calculated as:
A measure of the non-photochemical quenching co-efficient calculated as:
An alternative definition of non-photochemical quenching calculated as:
A measure of the electron transport rate calculated as:
* 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.
** 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.
*** 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.
$ 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.
$$ Horton, P., Ruban, A.V., Rees, D., Pascal, A. A., Noctor, G. and A. J. Young (1991). Control of the light-harvesting function of chloroplast membranes by aggregation of the LHCII chlorophyll-protein complexes. FEBS Lett., 292,1-4.
$$$ 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.