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Chlorophyll Fluorescence Overview

The capacity of a plant to carry out photochemistry is limited and will depend upon a range of factors including stresses caused by environmental conditions. Absorbed light energy in excess of that used for photochemistry must be effectively dissipated by non-photochemical processes. Such processes include the emission of heat and re-emission of small but diagnostically significant amounts of the absorbed radiation as longer wavelength red/far-red light energy. This re-emission of light is termed chlorophyll fluorescence. Although chlorophyll fluorescence emission from whole leaf systems is too weak to be viewed with the naked eye, it can be observed from illuminated extracts of a chlorophyll solution. Peak chlorophyll fluorescence occurs in the red region of the spectrum (685 nm) and extends into the infra-red region to around 800 nm.

Each of these processes operate in direct competition for a finite pool of absorbed energy, any change in energy utilization by one process produces a complementary change in the others. This fact enables chlorophyll fluorescence to be used as a rapid and reliable non-invasive probe of photochemistry. Specialist equipment is required for the analysis of the chlorophyll fluorescence signature. A Chlorophyll Fluorometer is designed specifically to detect the chlorophyll fluorescence emission from a sample. There are several different types of chlorophyll fluorometers available.

Hansatech Instruments design and manufacture chlorophyll fluorometers based around 2 different measurement techniques, Pulse Modulated Chlorophyll Fluorometers and Continuous Excitation or Prompt Chlorophyll Fluorometers. These chlorophyll fluorometers cover a wide range of applications in photosynthesis research programs. To learn more about these techniques and to determine which instrument best suits your application, please get in contact with PP Systems to speak to a products specialist. We look forward to being of assistance.

Pulse Modulated Fluorometers

FMS2 Field Fluorescence Monitoring System

Chlorophyll Fluorometer

The FMS2+ is a field portable pulse modulated chlorophyll fluorometer. The system is highly portable and fully self-contained.  A system of field interchangeable batteries provides power for a full day of fieldwork. A multi-charger system allows full recharge of the batteries overnight.

The FMS2+ control box now features a white LED lamp for actinic and saturation pulse intensities. It is fitted with a USB port to allow for simple connection to modern Windows® based computers (eliminating the need to use Serial to USB converter).

For information on the use of the FMS2+ with the CIRAS for simultaneous leaf gas exchange and chlorophyll fluorescence measurements, contact PP Systems.

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Product Features

  • 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

FMS1 Fluorescence Monitoring System

FMS1 Fluorescence Monitoring System for the lab from Hansatech Instruments

The FMS1 is a pulse modulated chlorophyll fluorometer operating from a mains power supply for laboratory use. The system may also be operated from an external 12V D.C. power supply or high capacity battery for near-field conditions. For information on the use of the FMS1 with the CIRAS for simultaneous leaf gas exchange and chlorophyll fluorescence measurements, contact PP Systems.

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Product Features

  • Pulse-modulated system
  • Integral LED & tungsten-halogen light sources
  • External device control interface
  • Programmable by Hansatech Scripting Language (HSL)
  • Optional leaf-clip with integral PAR/temperature sensor
  • Fiber optic for incorporation into O2 electrode units & IRGA
  • Windows® data acquisition & data analysis software

Continuous Excitation Systems

Pocket PEA

Pocket PEA Fluorometer from Hansatech Instruments

The Pocket PEA Chlorophyll Fluorimeter is suitable for teaching, research & a wide variety of commercial applications. The robust yet compact hand-held design provides ease of use & reliable operation.

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Product Features

  • Ultra-portable chlorophyll fluorometer
  • Rapid screening capability with single button operation & storage of up to 200 full data sets
  • Automatic calculation of parameters including Fv/Fm & OJIP analysis
  • Robust enclosure with sealed, high intensity optics
  • 100kHz sampling frequency with 16bit resolution
  • Bluetooth wireless data transfer as standard
  • Powerful Windows® data transfer & analysis software included

Handy PEA +

Advanced Continuous Excitation Chlorophyll Fluorimeter

Handy-PEA Plus is a compact, highly portable continuous-excitation type chlorophyll fluorescence analyzer. The rugged yet lightweight design makes Handy-PEA Plus particularly suitable for rapid, large scale screening of samples in the field or greenhouse in both research and teaching applications.

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Product Features

  • 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 Fmax
  • 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

M-PEA

MPEA Chlorophyll Fluorescence System from Hansatech Instruments

The M-PEA (Multi-Function Plant Efficiency Analyzer) combines high-quality fast fluorescence kinetic and P700+ absorbance studies with ground-breaking Delayed Fluorescence (DF) measurements providing one of the most comprehensive systems for the investigation of plant photosynthetic efficiency available.

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Fluorescence kinetics from M-PEA

Screenshot demonstrating prompt fluorescence and P700+ absorbance

Fluorescence kinetics from M-PEA

Screenshot demonstrating delayed fluorescence

Product Features

  • Advanced lab-based system for investigation of plant photosynthetic efficiency.
  • M-PEA-1 variant for prompt fluorescence and P700+ modulated absorbance measurements.
  • M-PEA-2 variant as M-PEA-1 with additional measurements of delayed fluorescence and leaf absorbtivity.
  • Sophisticated sensor unit with all optical emitters and detectors in robust, enclosed housing.
  • USB connection to a Windows® PC.
  • Comprehensive Windows® experimental design, data transfer and analysis software.

OJIP Parameters

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 quantifies the photosystem II behavior for (A) The specific energy fluxes (per reaction center) 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 centers 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)

Additional Resources

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.