M-PEA

Multi-function plant efficiency analyser

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

M-PEA Overview

M-PEA Multi-Function Plant Efficiency Analyser | Hansatech Instruments | Oxygen electrode and chlorophyll fluorescence measurement systems for cellular respiration and photosynthesis research

The M-PEA (Multi-Function Plant Efficiency Analyser) 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.

The M-PEA is a laboratory-based measurement system consisting of a control unit and sophisticated, robust sensor unit housing all optical emitters and detectors for all measurement elements.

The system is controlled from a comprehensive Windows® software package (M-PEA) which allows complex experiments to be designed, uploaded and executed by the M-PEA hardware. Recorded data is quickly downloaded to the software via a USB 2.0 connection.

The control unit is of convenient size with minimal footprint allowing measurements to be made in a busy lab environment where bench space is critical. The front panel consists of a power switch and indicator LED, optical sensor connection and a 4-line LCD display. The rear panel provides input for a 12V DC power supply and a USB 2.0 connection socket for interface to the M-PEA software running on a Windows® PC.

M-PEA Optical Sensor

M-PEA Multi-Function Plant Efficiency Analyser | Hansatech Instruments | Oxygen electrode and chlorophyll fluorescence measurement systems for cellular respiration and photosynthesis researchThe optical sensor unit is a robust enclosure designed to incorporate sophisticated electronics which effectively controls all of the light sources and detectors. The M-PEA-1 sensor unit includes a high-intensity red actinic source, a far-red light source, the prompt fluorescence detector and the modulated emitter/detector pair for P700+ absorbance measurements. M-PEA-2 additionally includes a high-sensitivity delayed fluorescence detector and a detector to measure leaf absorptivity.

All the optics are located behind a quartz window which seals the sensor unit providing effective protection for the optical assemblies against dust, dirt and moisture.

P700 Absorbance

P700 Absorbance Measurements | M-PEA Multi-Function Plant Efficiency Analyser | Hansatech Instruments | Oxygen electrode and chlorophyll fluorescence measurement systems for cellular respiration and photosynthesis research

The photosynthetic electron transport chain consists of 3 large protein complexes Photosystem II (PSII), Cytochrome (cyt b6/f), Photosystem I (PSI). P700 is the term used to describe the chlorophyll within the reaction center of PSI as this is the wavelength of light to which the photosystem is most reactive. Upon illumination using a strong light source, the photosynthetic electron transport chain is almost entirely reduced.

The electrons from this reduction, in turn, activate the enzyme ferredoxin-NADP+ reductase which leads eventually to NADP reduction and CO2 fixation. This initial reduction process is represented by the OJIP steps of the Kautsky induction curve during prompt fluorescence measurements.

The oxidation of P700 causes an increase in absorbance at wavelengths falling in the 800nm – 850nm band. M-PEA measures the transmission of P700 using a modulated LED with a peak wavelength of 820nm and a highly sensitive photodiode to monitor the absorbance of the PSI complex during prompt fluorescence measurements.

Since the 820nm LED is not actinic, M-PEA is able to use high light intensities without disturbing the PSII complex. Therefore, M-PEA presents a convenient, reliable method of measuring chlorophyll a fluorescence and transmission at 820nm simultaneously, thus allowing the study of the electron transport process during the Kautsky induction at both ends of the photosynthetic electron transport system.

M-PEA is also fitted with a far-red light source which can be used to preferentially excite the PSI complex. Re-reduction occurs via the intersystem electron transport chain by PSII activity, with electrons originating from hydrolysis.

The M-PEA uses an optically filtered, modulated 820nm LED for high-quality P700 absorbance measurements. P700 activity is recorded using an optimised low-noise, fast-response PIN photodiode and 16-bit A/D converter providing an excellent signal-to-noise ratio.

Delayed Fluorescence

M-PEA Delayed Fluorescence Measurements | M-PEA Multi-Function Plant Efficiency Analyser | Hansatech Instruments | Oxygen electrode and chlorophyll fluorescence measurement systems for cellular respiration and photosynthesis research

Delayed fluorescence (DF) has much in common with prompt fluorescence (PF) because it originates from the same chlorophyll molecules of the Photosystem II antenna complexes.

DF is essentially light that is emitted from green plants, algae and photosynthesising bacteria for a short time after they have been exposed to light, but after the prompt fluorescence emission has decayed. Delayed fluorescence occurs in the red/infrared region of the spectrum (the same as prompt chlorophyll fluorescence). However, the intensity of the delayed fluorescence emission is lower than that of prompt fluorescence by at least two orders of magnitude therefore requiring highly sensitive apparatus to measure the signal.

Like PF, the properties of the DF emission are highly sensitive to the functional state of Photosystem II and the photosynthetic reaction chain as a whole. Theoretically, DF bears even more information about the photosynthetic processes than PF. Still, a fluorescence measuring instrument can be found in almost every plant science research laboratory, while DF has not gained much popularity as a practical method to study photosynthetic organisms. One reason for such injustice is that DF is harder to register than PF. But the greatest difficulty in using DF is its interpretation, or extracting the valuable information from this extremely complex signal.

Fortunately, in recent years we’ve witnessed major advances both in development of electronic engineering and also in the theory of DF measurements. We are more and more able to utilise DF for practical scientific research. It is the combination of these 2 factors that has lead to the development of M-PEA.

The delayed fluorescence emission, natural to all green plants, has been known to scientists for over fifty years. It was first discovered by Strehler and Arnold (1951) when they were attempting to use firefly luminescence for the measurement of the light-induced accumulation of ATP in the green alga Chlorella. They found that even without the addition of luciferase and luciferin, there was a long-lived glow from algal cells and chloroplasts in darkness following illumination. The observed delayed fluorescence was characteristic of different photosynthesising samples used—leaves (Strehler and Arnold 1951), chloroplasts and photosynthesising bacteria (Arnold and Thompson 1956). Strehler and Arnold postulated that it was in fact chemiluminescence of the chlorophyll, caused by reversal of the photosynthetic reactions. The close relationship between DF and the photosynthetic reactions was confirmed undoubtedly in many studies and sometimes DF was found even more sensitive than the prompt fluorescence (Kramer and Crofts, 1996).

Leafclips & Sample Dark Adaptation

Pocket PEA & Handy PEA+ Leafclips (HPEA/LC, PPEA/LC) | Hansatech Instruments | Oxygen electrode and chlorophyll fluorescence measurement systems for cellular respiration and photosynthesis research

Continuous excitation fluorescence systems like Pocket PEA, Handy PEA+ and M-PEA rely on the use of a suitable leafclip system with 2 functions. Firstly, the leafclip shields the fluorescence detector from ambient light which would otherwise “blind” the sensor due to the comparatively high levels of red/infrared light within the same waveband as the fluorescence itself. Secondly, the leafclip pre-conditions or dark adapts a section of the sample prior to the measurement.

Any measurement of the maximum photochemical efficiency of Photosystem II (Fv/Fm) requires the sample to be fully dark adapted prior to measurement. During dark adaptation, all reaction centers within the sample are fully oxidised making them available for photochemistry and any latent 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.

Dark adaptation leafclips are constructed from plastic making them small and lightweight. The locating ring (which interfaces with the fluorimeter sensor) is positioned over the required area of the sample and has a central 4mm diameter hole which is covered using a shutter-plate. During measurement, this shutter slides back to expose the dark adapted sample to the focused LEDs and fluorescence detector. Pocket PEA leafclips have a black-coloured locating ring whereas Handy PEA+ and M-PEA leafclips have a white locating ring with a silvered underside which reflects incident light and minimises the build-up of heat on the sample. This ensures that the measurement is unaffected when measuring in high ambient light conditions.

M-PEA+ Software

M-PEA+ is a custom Windows® software package created for experimental design and deployment and comprehensive analysis of recorded data. M-PEA+ consists of 2 main elements:

M-PEA Protocol Editor

The protocol editor allows the creation of experiments for deployment on the M-PEA system. Experiments can range in complexity from a simple 1 – second prompt fluorescence measurement through to repeating, multi-flash measurements using prompt and delayed fluorescence, P700+ and relative absorptivity to probe the activity of PSI and PSII complexes within the photosynthetic apparatus.

Data Analysis Modules

Several different data presentation techniques have been combined in order to effectively demonstrate subtle differences in the fluorescence signature of samples. Data may be presented in graphical, tabulated or radial plots which can all be tailored to display any number of the 58 prompt fluorescence parameters measured by M-PEA. Transferred data may be exported to CSV format for further statistical analysis in external software packages.

M-PEA+ will run on all supported Microsoft® operating systems.

Common Continuous Excitation Fluorescence Parameters Measured

Common Continuous Excitation Fluorescence Parameters Measured | Hansatech Instruments | Oxygen electrode and chlorophyll fluorescence measurement systems for cellular respiration and photosynthesis research
Fo – Represents 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 Q_A is fully oxidised. This requires thorough dark adaptation.

Fm – The maximum fluorescence value obtained for a continuous light intensity. This parameter may only be termed as maximal if the light intensity used is fully saturating and the electron acceptor Q_A is fully reduced.

Fv – Indicates the variable component of the recording and relates to the maximum capacity for photochemical quenching. Calculated by subtracting the Fo value from the Fm value (Fm - Fo).

Fv/Fm – An indication of the maximum quantum efficiency of Photosystem II and widely considered to be a sensitive indicator of plant photosynthetic performance. Presented as a ratio between 0 and 1, healthy samples typically achieve 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 within PSII. Fv/Fm is presented as a ratio of variable fluorescence (Fv) over the maximum fluorescence value (Fm) and is calculated as (Fm - Fo)/Fv.

Tfm – Indicates the time at which the maximum fluorescence value (Fm) was reached. 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 Q_A on the reducing side of Photosystem II. If electron transfer from the reaction centers to the quinone pool is blocked (such as is the mode of action of the photosynthetically active herbicide DCMU), the area will be dramatically reduced.

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 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 (\Phi Eo = TRo/ABS)
  • Efficiency (\Psi o=Eto/Tro) with which a trapped exciton can move an electron into the electron transport chain further than Q_{A-}
  • 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 (PI) 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 a log scale. Therefore we say:

log PI = Driving~Force~DF

PI is derived according to the Nernst equation. It is the equation which describes the forces of redox reactions and general 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 Q_{A-}). 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 \times \Phi/(1-\Phi) \times \Psi/(1-\Psi)

or in fluorescence terms:

PI(abs) = ((dV/dto)/Vj) \times Fm/Fv \times (Fv/Fo) \times (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.

 

All Parameters Measured

  • OJIP data:
    • tFm
    • Area
    • Fo
    • Fm
    • Fv
  • Normalised data:
    • Fo/Fm
    • Fv/Fm
    • Fv/Fo
    • Vj = (Fj-Fo)/(Fm-Fo)
    • Vi = (Fi-Fo)/(Fm-Fo)
  • Specific fluxes:
    • ABS/RC
    • DIo/RC
    • TRo/RC
    • ETo/RC
    • REo/RC
  • Apparent fluxes per CSo:
    • ABS/RC
    • DIo/RC
    • TRo/RC
    • ETo/RC
    • REo/RC
  • Partial performances:
    • \Gamma(RC)/(1-\Gamma(RC))
    • \Phi(Po)/(1-\Phi(Po))
    • \Psi(Eo)/(1-\Psi(Eo))
    • PI(abs)
    • \Delta(Ro)/(1-\Delta(Ro))
  • Time marks:
    • Ft1
    • Ft2
    • Ft3
    • Ft4
    • Ft5
  • Partial areas:
    • Fo to Ft1
    • Ft1 to Ft3
    • Ft1 to Ft4
    • Ft1 to Ft5
    • Ft3 to Ft4
    • Ft4 to Ft5
    • Ft5 to Fm
  • Slopes & integrals:
    • dVg/dto
    • dV/dto
    • Sm = Area/Fv
    • N = Sm/Ss
    • Sm/tFm
  • Yield = flux ratios:
    • TRo/ABS = \Phi(Po)
    • ETo/TRo = \Psi(Eo)
    • ETo/ABS = \Phi(Eo)
    • REo/ETo = \Delta(Ro)
    • REo/ABS = \Phi(Ro)
  • Apparent fluxes per CSm:
    • (ABS/CSm)~Fm
    • DIo/CSm
    • TRo/CSm
    • ETo/CSm
    • REo/CSm
  • Total performance, driving force & rates:
    • PI(total)
    • DF(abs)
    • DF(total)
    • kP/ABS \times kF
    • kN/ABS \times kF
  • User parameter:
    • 3 User-entered values

System Components

M-PEA is supplied with the following components:

  • M-PEA control unit and sensor
  • HPEA/LC x 2: (20 Leafclips)
  • Mains power supply
  • Transit case
  • USB PC connection cable
  • USB Drive containing M-PEA+ software and manuals.

M-PEA Variant Feature Comparison

Signals Measured
Variant Prompt Fluorescence P700 Absorbance Delayed Fluorescence Relative Absorptivity
M-PEA-1
M-PEA-2

Technical Specifications

M-PEA Control Unit

  • Electronics:
    • 1 x high-performance 16-bit microcontroller
    • 1 x enhanced flash 8-bit controller
    • Dual channels:
      • 1 x modulated
      • 1 x non-modulated
    • 16-bit resolution A/D 10μs acquisition rate
    • Dual 16-bit D/A light source  controller
  • Memory: 32 Mb internal memory storage
  • Display: 4-line x 20-character LCD
  • Recording: Duration 0.001 seconds – 300 seconds (repeatable up to 100 x per protocol)
  • Comms: USB 2.0 full speed (12 Mb/s)
  • Power: 12V DC @ 1 amp
  • Dimensions: 230mm (w) x 190mm (d) x 85mm (h). 1.4kg

M-PEA-1 Optical Sensor Unit

  • Illumination:
    • Actinic:
      • Focused ultra-bright LED with NIR short- pass cut-off filter
      • Dominant λ625nm
      • Spectral half-width 20nm
      • Max intensity 5,000 μmol m-2 s-1.
    • Far-red:
      • Focused ultra-bright LED with long-pass filter
      • Max intensity >1,000 μmol m-2 s-1
    • P700:
      • Optically filtered pulse-modulated 820nm LED
      • Intensity 0% – 100% in 1% steps
  • Detectors:
    • PF: Low-noise, fast-response PIN photodiode with 730nm (± 15nm) bandpass filter
    • P700: Low-noise, fast-response PIN photodiode with an optical bandpass filter.

M-PEA-2 Optical Sensor Unit

  • Illumination: Sources as in M-PEA-1 sensor unit
  • Detectors: As M-PEA-1 but with additional detectors:
    • Delayed fluorescence: High-sensitivity wideband avalanche photodiode with 7 30nm (± 15nm) bandpass filter
    • Leaf absorptivity: Low-noise, fast-response PIN photodiode

Publications

One of the extensions of the Google search facility is Google Scholar. It allows you to search through vast archives of peer-reviewed published papers and journals that have been posted online.

Use the tool below to enter search terms as required. As an example, hansatech instruments M-PEA has already been entered into the search box. Press the "Search" button to view the Google Scholar results for this search string.

Admittedly, some of the results link to journals which require subscription in order to view the publications but even so, we have found this facility to be a valuable tool.

Google Scholar

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