Continuous Excitation Chlorophyll Fluorescence Measurement
General Parameters Measured
Fo
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.
Fm
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.
Fv
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
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
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.
Area
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 ( Abs/RC)
- Trapping (TRo/RC)
- Dissipation (DIo/CS)
- Electron transport (ETo/RC)
and (B) the flux ratios or yields:
- Maximum yield of primary photochemistry (φEo = TRo/ABS)
- Efficiency (Ψo=Eto/Tro) with which a trapped exciton can move an electron into the electron transport chain further than QA-
- Quantum yield of electron transport (Eto/CS)
The concentration of active PSII reaction centres per excited cross section (RC/CS) 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:
log PI = Driving Force DF
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:-
Potential = log x/(1-x)
where x is the fraction of a partner in the reaction A to B. Therefore:
X = A /(A + B)
and if you now convert to:
X/(1-X) = A / B
or for redox reactions
log (red)/(ox)
Now the total potential in a mixture is the sum of the individual potentials or:
Potential total = log X1/(1-X1) + log X2/(1-X2) ....,etc
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
X1 = RC Chlorophyll per total chlorophyll = CHL(RC)/CHL(total)
therefore:
X1/(1-X1) = CHL(RC) / ( CHL(tot) - CHL(RC)) = CHL(RC) / CHL(antenna) = RC/ABS
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:
PHI(Po) = maxTrapping / Absorption = TRo/ABS = Fv/Fm
The Driving force of the light reactions is therefore:
DF(PHI(Po)) = log PHI/(1 - PHI) = log (Fv/Fm) / ( 1 - Fv/Fm) = log Fv/Fo = log kP/kN
- 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:-
psi(o) = ETo/TRo = (1 - Vj)
Where Vj = relative variable fluorescence at 2 ms or at the step J therefore:
Vj = (Fj - Fo)/(Fm - Fo) and psi(o) = 1 - Vj = (Fm - Fj) / (Fm - Fo)
Therefore the force of the dark reactions is:
DF(psi) = log psi/(1-psi) = log (1-Vj)/Vj
Now all three components together make:
DF (total on a chl basis) = DF(RC) + DF(phi) + DF(psi)
or without log
PI(abs) = RC/ABS x PHI/(1-PHI) x psi/(1-psi)
or in fluorescence terms:
PI(abs) = ((dV/dto)/Vj) x Fm/Fv x (Fv/Fo) x (Fm-Fj)/(Fj-Fo)
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.