Advanced Continuous Excitation Chlorophyll Fluorimeter
- Compact (170 x 85 x 40mm), lightweight (565gms)
- Large-scale screening capacity up to 1000 full trace data files
- High time resolution detection for discrimination of fast chlorophyll fluorescence induction kinetics
- Saturating high intensity focused LED array for accurate determination of Fm
- Upload user-defined, repeatable protocols for automatic field execution
- Interchangeable sensor unit cables with lengths of up to 20 metres
- Powerful Windows® data transfer & analysis software included
The Handy PEA chlorophyll fluorimeter consists of a compact, light-weight control unit encapsulating sophisticated electronics providing the high time resolution essential in performing measurements of fast chlorophyll fluorescence induction kinetics.
The chlorophyll fluorescence signal received by the sensor head during recording is digitised within the Handy PEA control unit using a fast Analogue/Digital converter. The chlorophyll fluorescence signal is digitised at different rates dependent upon the different phases of the induction kinetic. Initially, data is sampled at 10µs intervals for the first 300µseconds.
Simple to configure and operate, the Handy PEA chlorophyll fluorimeter features the capacity to store up to 5 user-defined protocols for different field applications. Protocols are written using a custom Windows® software package, PEA Plus (supplied). This allows single or multiple measurement assays with optional pre-illumination periods to be defined and uploaded to the memory of Handy PEA via RS232 serial communications. The use of protocols ensures maximum reproducibility of results during field applications involving large scale screening away from a laboratory environment. A waterproof, tactile keypad allows selections and inputs to be made and a liquid crystal display module presents menu options and data.
Up to 1000 recordings of between 0.1 – 300 seconds may be saved in the memory of Handy PEA chlorophyll fluorimeter. Saved data may be viewed onscreen in numerical format with calculated parameters or transferred to the PEA Plus software where it may be viewed graphically or exported to external software packages for further statistical analysis.
The sensor unit consists of an array of 3 ultra-bright red LED’s optically filtered to a peak wavelength of 650 nm, which is readily absorbed by the chloroplasts of the leaf, at a maximum intensity of up to 3500 µmol m-2 s-1 at the sample surface. The LED’s are focused via lenses onto the leaf surface to provide even illumination over the area of leaf exposed by the leafclip (4mm dia). LED’s have the advantage of being rugged, emitting low levels of heat, and of rising to full intensity very rapidly (typically microseconds) after being switched on. This feature eliminates the inaccuracies of Fo measurement and the constraints on speed and reliability associated with a shutter which is a necessary item in systems using filament lamps rather than LED’s.
An optical feedback circuit monitors and corrects changes in the output intensity of the LED’s. These changes are caused by internal heat build up in the LED’s. The circuit also compensates for intensity changes caused by variation in ambient temperature. The light source is calibrated before leaving the factory but may be calibrated by the user at regular intervals using the SQS Serial Quantum Sensor.
The detector is a high performance Pin photodiode and associated amplifier circuit. The optical design and filtering ensure that it responds maximally to the longer wavelength fluorescence signal and blocks the reflected shorter wavelength LED light used as the source of illumination.
The sensor unit is connected to the Handy PEA control unit via a standard connection cable of 1m in length however, connection cables of up to 10m in length are also available on request.
Leafclips and Sample Dark Adaptation
The first step in the measurement process using the chlorophyll fluorimeter is to cover the sample area to be analysed, with a small, lightweight leafclip. The clip 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 fluorimeter optical assembly is constructed from black plastic. This ensures that the measurement is unaffected when measuring during conditions of high ambient light intensity.
The leaf or needle rests on a foam pad whilst in the clip in order to minimise damage to the structure of the leaf. 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. Some users even make measurements at night, thus ensuring an adequate supply of readily dark adapted samples and zero waiting time!
PEA Plus & PEA Plus Mobile Software
PEA Plus provides a comprehensive tool for in-depth analysis of data recorded by the chlorophyll fluorimeter.
Several different data presentation techniques have been combined in order to effectively demonstrate subtle differences in the fluorescence signature of samples which could be indicative of stress factors affecting the photosynthetic efficiency of the plant.
A suitable IPAQ/PDA running Windows Mobile 5.0 or 6.0® may be used in the field for data storage and limited review of parameters and traces using the PEA Plus Mobile software. Records are downloaded via Bluetooth wireless communication.
Common Parameters Measured
The Fo parameter is thought to represent emission by excited chlorophyll a molecules in the antennae structure of Photosystem II. The true Fo level is only observed when the first stable electron acceptor of Photosystem II called Qa is fully oxidised. This requires thorough dark adaptation. Fo occurs at time base 0. It is the almost instantaneous (nanoseconds range) rise to an origin level of chlorophyll fluorescence upon illumination using a chlorophyll fluorimeter. Due to restrictions in electronics technology and the speed of fluorescence detection, it is not possible to measure the true Fo. However, it is possible to estimate the Fo level to a high degree of accuracy using a mathematical algorithm.
This is the maximum chlorophyll fluorescence value obtained for a continuous light intensity. This parameter may only be termed as maximum fluorescence if the light intensity provided by the chlorophyll fluorimeter is fully saturating for the plant and the electron acceptor Qa is fully reduced. If the light intensity used for the recording is not sufficiently high, the plant may not be fully saturated in all circumstances. The peak fluorescence level (Fp) achieved in these circumstances would not be maximal and therefore should not be used as Fm. Consequently the ratio Fv/Fm would not be correct and the ratio would strictly be Fv/Fp with a reduced value. This was commonly the case when using an older chlorophyll fluorimeter with a lower maximum light intensity for excitation due to constraints in technology. Rapid advances in LED technology allow modern day analytical instrumentation to be designed to incorporate ultra-bright LED’s providing fully saturating light intensities in smaller, more manageable units such as the Handy PEA, Pocket PEA and M-PEA fluorimeters.
The Fv parameter indicates the variable component of the recording and relates to the maximum capacity for photochemical quenching. It is calculated by subtracting the Fo value from the Fm value.
Fv/Fm is a parameter widely used to indicate the maximum quantum efficiency of Photosystem II. This parameter is widely considered to be a sensitive indication of plant photosynthetic performance with healthy samples typically achieving a maximum Fv/Fm value of approx. 0.85. Values lower than this will be observed if a sample has been exposed to some type of biotic or abiotic stress factor which has reduced the capacity for photochemical quenching of energy within PSII. Fv/Fm is presented as a ratio of variable fluorescence (Fv) over the maximum fluorescence value (Fm).
Tfm is a parameter used to indicate the time at which the maximum fluorescence value (Fm) was reached. This parameter may be used to indicate sample stress which causes the Fm to be reached much earlier than expected.
The area above the fluorescence curve between Fo and Fm is proportional to the pool size of the electron acceptors Qa on the reducing side of Photosystem II. If electron transfer from the reaction centres to the quinone pool is blocked such as is the mode of action of the photosynthetically active herbicide DCMU, this area will be dramatically reduced.
The Area measurement is a very useful parameter as it highlights any change in the shape of the induction kinetic between Fo and Fm which would not be evident from the other parameters e.g. Fo, Fm, Fv/Fm which only express changes of amplitude of the extreme Fo and Fm. An example of its use would be following the time dependence of herbicide penetration into the leaf by following changes in the induction kinetic with time.
Time Marks Parameters
The PEA Plus and M-PEA Plus software packages extract chlorophyll fluorescence values from the recorded data from Handy PEA, Pocket PEA and M-PEA chlorophyll fluorimeters at 5 pre-defined Time Marks. The times are:
- T1 = 50 microseconds
- T2 = 100 microseconds
- T3 = (K step) 300 microseconds
- T4 = (J step) 2 milliseconds
- T5 = (I step) 30 milliseconds
Chlorophyll fluorescence values at these Time Marks are used to derive a series of further biophysical parameters, all referring to time base 0 (onset of fluorescence induction), that quantify the photosystem II behaviour for (A) The specific energy fluxes (per reaction centre) for:
- Absorption ()
- Trapping ()
- Dissipation ()
- Electron transport ()
and (B) the flux ratios or yields:
- Maximum yield of primary photochemistry ()
- Efficiency () with which a trapped exciton can move an electron into the electron transport chain further than QA-
- Quantum yield of electron transport ()
The concentration of active PSII reaction centres per excited cross section () is also calculated.
Performance Index Parameters (OJIP Analysis)
The Performance Index is essentially an indicator of sample vitality. It is an overall expression indicating a kind of internal force of the sample to resist constraints from outside. It is a Force in the same way that redox potential in a mixture of redox couples is a force. Exactly the PI is a force if used on log scale. Therefore we say:
The PI or Performance Index is derived according to the Nernst equation. It is the equation which describes the forces of redox reactions and generally movements of Gibbs free Energy in biochemical systems. Such a force (or potential = force) is defined as:-
where x is the fraction of a partner in the reaction A to B. Therefore:
and if you now convert to:
or for redox reactions
Now the total potential in a mixture is the sum of the individual potentials or:
In our case PI (on an absorption basis or on a chlorophyll basis) has three components:
The first component shows the force due to the concentration of active reaction centers
RC/ABS is a parameter of the JIP test and it is related to the force generated by the RC concentration per antenna chlorophyll.
The second component is the force of the light reactions, which is related to the quantum yield of primary photochemistry:
The Driving force of the light reactions is therefore:
The third component is the force related to the dark reactions (after Qa-). These are normal redox reactions in the dark.Expressed by the JIP-test as:
Where = relative variable fluorescence at 2 ms or at the step J therefore:
Therefore the force of the dark reactions is:
Now all three components together make:
or without log
or in fluorescence terms:
A more detailed derivation and explanation is beyond the scope and intention of this web page. Further detailed information may be obtained from the following publications which may be downloaded as PDF documents from the following links.
R.J. Strasser, A. Srivastava and M. Tsimilli-Michael The fluorescence transient as a tool to characterize and screen photosynthetic samples.
Strasser, R.J., M. Tsimilli-Michael and Srivastava, A. Analysis of the Fluorescence Transient.