The New Standard in
Portable Fluorometers
Chlorophyll Fluorescence

Chlorophyll Fluorescence and Definitions

The following is a generalized description of the photosynthesis light reaction and the value of chlorophyll fluorescence in photosystem II. For more specific information please refer to papers cited in this discussion or contact Opti-Sciences for more information.

Light energy utilized in photosynthesis by higher plants and algae cells is collected first by an antenna pigment system and transferred to reaction centers where light quanta are converted to chemical energy by chlorophylls in a protein environment. Electron transfer takes place in the reaction center when a chlorophyll molecule transfers an electron to a neighboring pigment molecule. Pigments and protein involved in this primary electron transfer define the reaction center. This initial electron transfer is also called charge separation.

Competing models of energy capture and transfer exist. In the “puddle model” of Photosystem PS II each reaction center possesses its own independent antenna system. In the “lake model” of Photo-system PS II reaction centers are connected by shared antenna. The “lake model” is considered more realistic for terrestrial plants.

Reaction centers are of two types, Photosystem II (PSII), and Photosystem I (PSI). both are located in the thylakoid membrane of a chloroplast of higher plants. In bacteria they are in a membrane surrounding the cytoplasm or in more intricate constructs. All plants the produce oxygen have both types of reaction centers.

It is in PSII that oxygen evolution and the splitting of water occurs. To reduce the PSII reaction center an electron is pulled from the water splitting complex.

After charge separation, electrons flow to other nearby plastoquinone molecules in the thylakoid membrane by oxidation reduction reactions. They act as energy transfer molecules in electron transport chain.

The next stop is a cytochrome b6f complex. Eventually this complex supplies an electron to reduce PSI. PSI then goes through a somewhat similar process to PSII, and eventually produces NADPH.

In addition, protons created from PSII functions, the splitting of water, and the reaction in the cytochrome b6f complex, enter the thylakoid lumen and are used by an ATP pump in the thylakoid membrane in the presence of ATP synthase, to create ATP from ADP. Both ATP and NADPH are used as energy sources to drive the Calvin Benson Cycle of carbon fixation.

PSI goes through a similar process to PSII, however, the low fluorescent signal of PSI does not vary with plant stress or with various photosynthetic functions as it does with PSII. Therefore PSII is of primary interest. Factors such as light levels, light quality, water availability, nutrient availability, heat, cold, herbicides, pesticides, pollution, heavy metals, disease, and genetic make up can all have an impact on CO2 assimilation, plant health and condition. They also are reflected in the fluorescence signal in PSII. Therefore, by using a chlorophyll fluorometer one can quantify the impact of these factors on plants to improve breeding and production programs, and to better understand plant functions.

The most prominent pigments that absorb this energy are chlorophyll-a and chlorophyll-b. Other accessory pigments may be involved such as carotenoids, or phycobilins in cyanobacteria, or bacteriochlorophyll in some bacteria.

Light energy absorbed initially by the antenna and transferred to the reaction centers is channeled by a number of different processes including photochemistry, photo-protective heat dissipation, other heat dissipation and about 3%-9% of the light energy absorbed by chlorophyll pigments is re-emitted as fluorescence. The emission peak is of a longer wavelength than the excitation energy. This effect was first observed more than 100 years ago, when N.J.C. Müller (1874) by visually using colored glass filters. He also noted that fluorescence changes that occur in green leaves are correlated with photosynthetic assimilation. Lack of appropriate technical equipment prevented a more detailed investigation of this phenomenon. The light energy drives photosynthetic electron transport through PSII and PSI leading to the oxidation of water, oxygen evolution, the reduction of NADP+ to NADPH, membrane proton transport and ATP synthesis.

The loss of light energy from the reaction center as fluorescence comes primarily from the PSII reaction. When the chloroplast or leaves have been dark-adapted, the pools of oxidation-reduction intermediates for the electron transport pathway return to an oxidized state. Upon illumination of a dark-adapted leaf, there is a rapid rise in fluorescent light emission from PSII followed by a decrease to steady state fluorescence as CO2 fixation starts to occur. Changes in the intensity of the fluorescence emission are a sensitive reflection of changes in the photosynthetic apparatus. Following many years of study, chlorophyll fluorescence has been shown to represent changes in the health and function of the photosynthetic process. This includes all the reactions from the oxidation of water, charge separation, electron transport, development of the electrochemical gradient, the photo-protective mechanisms of the xanthophyll cycle, and the change in ph of the thylakoid lumen. Even changes in the plant that affect stoma opening and gas exchange with the atmosphere are reflected by changes in the fluorescence characteristics of a leaf. Vast amounts of research have shown excellent correlation between carbon fixation and chlorophyll fluorescence data.

This is a typical Dark-Light kinetic pulse modulated trace.
Definitions are described below.

Figure 1-1 • Dark–Light Pulse modulated Chlorophyll Fluorescence Trace

Definitions:

Dark adapted

– This is a term that means that the area of the plant, or the entire plant, to be measured has been in the dark for an extended period of time before measurement. Dark adaption requirements may vary for dark adapted tests. Dark adaption times of ten minutes to 60 minutes are common, and some researchers use pre-dawn values. Dark adapted measurements include Fv/Fm, OJIP measurements, and most non-photochemical quenching parameters. Fv/Fm and most OJIP measurements take only a second to make a measurement. Non-photochemical quenching parameters take longer.

Light adapted

– Yield measurements are made when a sample is already exposed to existing light conditions. No dark adaption is required. When a sample is exposed to light it normally takes several second to a few minutes for photosynthesis to reach steady state. To obtain a reliable yield measurement photosynthesis must reach steady state. This is not a concern when using ambient sunlight or artificial greenhouse light however clouds and light fleks below a canopy level can cause problems. If one uses a built in fluorometer illuminator to measure yield make sure that steady state photosynthesis has been reached. This is normally a very fast test that take a second or two and it has the added advantage that it does not require dark adaption. Light adapted measurements include Yield or ΔF/Fm’.

PAR

– Photosynthetically Active Radiation between 400nm and 700nm. Measured in either µmls or µE The Term PAR means photosynthetically active radiation in the wave band between 400-700 nm. PAR can be measured in different dimensions such as Watts per meter or in micro- Einsteins or micro-moles. When using a PAR Clip, dimensions will always be in the equivalent terms, micro-Einsteins, or micro-moles

PPFD

- Photosynthetic Photon Flux Density is the photon flux density of PAR. Measured in either or. PPFD, or “photosynthetic photon flux density”, is the number of PAR photons incident on a surface in time and area dimensions. These terms are equivalent for PAR Clip leaf radiation measurements. Furthermore, both can be presented in either of the equivalent dimensions, micro-moles (µmls) or micro-Einsteins (µE).

µmls

- is a micro mol (also abbreviated µmol. This a dimension that involves both time and area. It is the equivalent to the micro Einstein. Both terms have been used extensively in biology and radiation measurements.

µE

– is a micro Einstein This a dimension that involves both time and area. It is the equivalent to the micro mol. Both terms have been used extensively in biology and radiation measurements.

1 µE = 1 µmol m-2 s-1 = 6.022 x1023 photons m-2 s-1

Actinic Light source

– This is any light source that drives photosynthesis. It may be the Sun, or an artificial light. Higher end fluorometers contain one or more built-in artificial actinic light sources for experimentation with specific repeatable radiation (or light) levels.

Saturation pulse

– is short pulse of very intense light designed to fully reduce a leaf's PSII system. It is created by an artificial light source in all modulated fluorometers.

Far red light

– is a light source that provides light above 730 nm to allow the transfer of electrons to PSI and allow the rapid re-oxidation of PSII.

Modulated light Source

– This is the measuring light source in a modulated fluorometer. It is a precision light diode that is usually either red or blue. Most fluorometers usually have one or the other. The OS5p has both. They use a very low intensity, usually between 0.2 µmls and 1µml, to allow measurement without driving photosynthesis.

Fo

– is the dark adapted initial minimum fluorescence.

Fm

– is maximal fluorescence measured during the first saturation pulse after dark adaption.

Fs

– also known as F’ is the fluorescence level related to the actinic light and is a reflection of level of photosynthetic activity.

Fms

– also known as Fm’ is the saturation pulse value that is not dark adapted. They are lowered values due to NPQ or non-photochemical quenching. When this parameter has reached steady state, it is used to calculate photosynthetic Yield along with Fs. Fms at steady state is also used to calculate qN, NPQ, qP, qL, Y(NPQ), Y(NO), qE, qT, and qI.

Fod

– also known as Fo’ is the minimal value after the far red light is turned on for several seconds after the actinic light has been turned off. It represents Fo with non-photochemical quenching.

Ft

– is the current instantaneous fluorescent signal.

Fv/Fm

= (Fm – Fo) / Fm This is a dark adapted test used to determine Maximum quantum yield. This ratio is an estimate of the maximum portion of absorbed quanta used in PSII reaction centers. It is important to properly dark adapt samples for this test. Fo will be raise and Fm will be lowered if dark adaption is inadequate. Since dark adaption requirement can vary with species, varieties, mutants, and sun vs. shade leaves testing should be done to ensure proper dark adaption. (Kitajima and Butler, 1975)

Y

= (Fms – Fs) / Fms This test is also known as ΔF/Fm’. Yield of PSII is a light adapted test normally taken at steady state photosynthesis levels. It provides a measure of actual or effective quantum yield. This ratio is an estimate of the effective portion of absorbed quanta used in PSII reaction centers. (Genty, 1989)

Quenching Parameters

Quenching parameters allow the quantification of both the effective photochemical state of the PSII regarding the fraction of PSII centers that remain open or oxidized at any time, and the non-photochemical photosynthetic mechanisms involved in photo-protection, state 1 and state 2 transition quenching, photo-inhibitor and photo-damage.

Puddle Model and Lake Model of Antennae

In the “puddle model” of Photo-system PS II each reaction center possesses its own independent antenna system. In the “lake model” of Photo-system PS II reaction centers are connected by shared antenna. The lake model has proven to be the more realistic model for terrestrial plants. Research has shown that some marine species have a distinct antenna architecture. With this in mind, some of the quenching parameters of the “puddle model” are incompatible with those of the “lake model”. (David M. Kramer, Giles Johnson, Olavi Kiirats & Gerald E. Edwards 2004)

qP = (Fm' - F) / (Fm' - Fo')

NPQ = (Fm - Fm') / Fm'

NPQ = qE + Qt + qI

qN = (Fm - Fm') / (Fm - Fo)

qL = qP(Fo'/F')

Y(NO) = 1/NPQ + 1 + qL((Fm/Fo)-1)

Y(NPQ) = 1 - Y - Y(NO)

Understanding the graphs above:

In each case the sample is first dark adapted. The test is started and Fo, or minimal fluorescence, is measured without actinic light. Then a saturation pulse occurs and completely closes all receptors in PSII by completely reducing PSII. Fm, maximal fluorescence, is the result. After the saturation pulse, an actinic light is turned on and the fluorescent signal declines slowly with the onset of CO2 fixation until it reaches steady state. Photochemical quenching a measure of open PSII centers, photo-protective non-photochemical quenching and other heat dissipation mechanisms occur. Saturation pulses during steady state photosynthesis provide Fm', maximal fluorescence in this situation after NPQ has reached equilibrium with photochemistry. qP, or qL in the lake model, now represents the fraction of PSII receptors that remain open or oxidized. F (or Fs) represents fluorescence related to current steady state photochemical levels. At the point where the actinic light is turned off, far red illumination is turned on to allow the transfer of electrons quickly to reduce PSI, and allow the re-oxidation of PSII. Fo' represents this value with un-relaxed non-photochemical quenching. The rising values of the saturation pulses after the actinic light has been turned off represent the relaxation of NPQ over time. A portion of NPQ, qE (or Y(NPQ) in the lake model), represents photo-protection mechanisms of thylakoid lumen ph and the xanthophyll cycle. The remainder of NPQ represents qT, and qI, (or Y(NO) in the lake model). qT is quenching due to state 1 and state 2 transitions and is negligible in higher plants. qI represent photo-inhibition and photo-damage.

Definitions for Quenching Parameters:

NPQ

is non-photochemical quenching and is a measure of heat dissipation and a combined total for the combination of photo-protective mechanisms, state 1 and state 2 transition quenching, and photo-inhibition and photo-damage. NPQ = qE + qT + qI. NPQ is an alternate expression of non-photochemical quenching. It provides an estimate of quenching without knowledge of Fo. The advantage of NPQ over qN depends on the specific application. NPQ is more heavily affected by non-photochemical quenching that reflects heat-dissipation of excitation energy in the antenna system. So it may be thought of as an indicator of 'excess excitation energy'. Alternatively, NPQ is relatively insensitive to the part of non-photochemical quenching associated with qN values lower than 0.6 This range of qN is affected by ΔPH of the thylakoid lumen which is an important aspect of photosynthetic regulation. (Bilger & Björkman, 1990)

qN

is similar to NPQ but requires Fod or Fo’ in the calculation. qN is defined as the coefficient of non-photochemical fluorescence quenching. The original definition of this term implied that fluorescence quenching affects primarily the 'variable fluorescence' (Fv) and not the minimal fluorescence (Fo). In cases where qN is greater than 0.4 this may not be a good assumption. This will affect the calculated qN value. By using the Far-Red source after actinic illumination, the PSII acceptors re-oxidized and PSI is reduced. A new Fod value is measured and used for corrections to the quenching coefficients. (puddle model) (Van Kooten & Snel, 1990)

qE

is the quenching parameter that represents the photo-protective mechanisms in the leaf that allow rapid compensation for changes in light levels due to cloud cover and increased light intensity. It is directly related to Δph of the thylakoid lumen and the xanthophyll cycle. (puddle model) (Muller P., Xiao-Ping L., Niyogi K. 2001)

qT

is the quenching parameter that represents state 1 and state 2 transitions. This value is negligible in higher plants. (puddle model) (Muller P., Xiao-Ping L., Niyogi K. 2001)

qI

is the quenching parameter that represents photo-inhibition and photo-damage. (puddle model) (Muller P., Xiao-Ping L., Niyogi K. 2001)

Y(NPQ)

is a lake model quenching parameter that represents heat dissipation related to all photo-protective mechanisms. (David M. Kramer, Giles Johnson, Olavi Kiirats & Gerald E. Edwards 2004)

Y(NO)

is a lake model quenching parameter that represents all other components of non-photochemical quenching that are not photo-protective. (David M. Kramer, Giles Johnson, Olavi Kiirats & Gerald E. Edwards 2004)

qP

is the quenching parameter that represents photochemical quenching. It is a measure of the fraction of still open PSII reaction centers. qP is defined as the coefficients of photochemical fluorescence quenching. The original definition of this term implied that fluorescence quenching affects primarily the 'variable fluorescence' (Fv) and not the minimal fluorescence (Fo). In cases where qN is greater than 0.4 this may not be a good assumption. This will affect the calculated qN and qP values. By using the Far-Red source for post illumination, the PSII acceptors may be re-oxidized through the illuminations affect on PSI. A new Fod value may be measured and used for corrections to the quenching coefficients. This assumes the PSI acceptors are properly activated, which may not be the case in a dark adapted sample. therefore the Fod determination should be done after induction of photosynthesis has been done for several minutes. (puddle model) (Van Kooten & Snel, 1990)

qL

is the lake model quenching parameter that represents photochemical quenching. It is a measure of the fraction of still open PSII reaction centers. (David M. Kramer, Giles Johnson, Olavi Kiirats & Gerald E. Edwards 2004)

Equations for quenching parameters:

qP

= (Fm’- F) / (Fm’-Fo’)

NPQ

= (Fm - Fm’) / (Fm’)

NPQ

= qE + qT + qI

qE

= Fm’ after rapid relaxation is complete with the actinic light turned off usually one to ten minutes - Fm’ during steady state fluorescence with actinic light on/Fm’ at steady state.

qT

= Fm’ after rapid relaxation is complete usually with the actinic light turned off usually one hour - Fm’ at qE /Fm’ at steady state.

qI

= Fm-Fm’ at qT/ Fm’ at steady state.

qN

= Fm - Fm’/ Fm-Fo

qL

= qP(Fo’/F’)

Y(NO)

= 1/NPQ +1 + qL((Fm/Fo)-1)

Y(NPQ)

= 1 - Y - Y(NO)

1

= qL + Y(NPQ) + Y(NO)

Relative Electron Transport Rate

Relative Electron Transport Rate, written ETR

µmls = (Y) (PAR) (.84) (.5) = (quantum yield of PSII) (measured photosynthetically active radiation measured in uMols quanta m-2 s-1.)( leaf absorption coefficient)(fraction of absorbed light by PSII antennae). ETR is valuable for many types of plant stress investigations. This relative form of ETR provides a great deal of information as yield varies with PAR radiation (light intensity). For more information see the stress guide listed on this web site.

Absolute electron transport rate is measured by CO2 gas exchange measurements.

Relative ETR does not correlate exactly because while most of radiation is absorbed in the upper layers and provide fluorescent information, some radiation does enter lower layers and the information is not captured in fluorometry. CO2 gas exchange ETR includes information from deeper layers. (U. Schreiber 2004).

Nitrogen deficiency Test (FRFex360/FRFex440)

This Test is available with the purchase of the Universal Par clip.

Nitrogen Stress in plants can be determined by the ratio of UV excited and blue excited far red fluorescence, (FRFex360/FRFex440). Unlike leaf absorption techniques used for nitrogen testing, nitrogen stress can be distinguished from sulfur stress with this measurement (Sampson 2000). FRFex360/FRFex440 measures the concentration of UV absorbing compounds in the leaf epidermis. Based on the Carbon/nutrient balance hypothesis, excess fixed carbon relative to nutrient uptake stimulates the Shikimate acid pathway. This creates production of phenolic and other carbon based compounds that reside primarily in the epidermis (Cerovic 1999), (Price 1989), (Waterman and Mole 1994). A decrease of this ratio indicates higher concentrations of phenolic compounds due to nitrogen stress. These compounds absorb UV light and cause a decrease in excitation of chlorophyll molecules in the mesophyll. The blue light fluorescence acts as a measuring standard because it passes through the epidermis unaffected by these compounds. Therefore a ratio of unabsorbed UV light far red fluorescence to unabsorbed blue light far red fluorescence provides a sensitive test for nitrogen deficiency. It was found by Sampson (Sampson 2000) that sulfur deficiency did not affect the ratio.

The nitrogen test is a fast test taking less than two second. One thousand measurements are made and the average value is reported. The test can be done with steady state fluorescence.

With this test field plants will measure lower ratios than green house plants and older leaves will measure lower ratios than newer leaves due to the creation of some flavenoids by solar UV exposure. Controlling these variables is important. One should choose the same leaf and leaf age on plants to be measured. A plant that is without nitrogen stress can be used as a standard to set the ratio at one. Take care to use a field plant for field tests, and a green house plant for green house tests.

OJIP stress testing

OJIP is a fast, dark adapted test protocol that uses a high signal capture rate for analysis of fluorescence changes with emphasis on the initial fluorescence rapid rise kinetics using strong actinic light. In the initial fluorescence kinetic rise it has been found that the resulting curve displays intermediate peak values before reaching Fm, or P, maximum Fluorescence. These intermediate peaks or steps are designated J, I, and P with O being the initial measured fluorescence signal value after 20 µsec (Strasser 2004).

Besides the JIP steps an additional step called the K step appears during specific types of stress. (R. J. Strasser 2004).

Stress identification example: The K step = F270µs (a fluorescence step that appears 270 microseconds after the actinic light is turned on) in the OKJIP test. The K peak is activated when the OEC, or oxygen evolution cycle, is affected. Research has shown that heat stress, nitrogen stress, and water stress can activate the K peak (R. J. Strasser 2004). The performance index parameter captures this information.

Performance index: PI

Probably the most used parameter in the OJIP test is performance index: PI. Stresses that do not directly affect PSII do not cause a decrease of the Fv/Fm-value. PI was developed in an effort to solve that problem (G. Schansker 2008).

Performance index, PI, is an indicator of three main attributes, which determine potential photosynthetic activity; reaction centers density, probability that an absorbed photon is used for a charge separation, and forward electron transfer. The idea is that if a stress will affect any of these components, the effect will show up in the performance index (G. Schansker http://come.to/bionrj). In other words, on a less mechanistic level, besides capturing Fm, and Fo, information as found in Fv/Fm, PI also incorporates O and J information and the initial slope of O-K.

Measured and calulated OJIP Values with direct readout

  1. Ft

    = Fluorescence at the onset of actinic illumination
  2. F20µs

    = F20µs; fluorescence intensity at 20 µs =

    O location

  3. F70µs

    = fluorescence at 70µs
  4. F270µs

    = fluorescence at

    270µs =step K

  5. Fj

    = fluorescence intensity at

    J-step

    (at 2 ms)
  6. Fi

    = fluorescence intensity at

    I-step

    (at 60 ms)
  7. FP

    = maximal fluorescence intensity = Fm =

    P-step

  8. tFm

    = time to reach maximum fluorescence intensity
  9. Area

    = area above the fluorescence curve to Fm (or P). If area decreases compared to a non stressed plant, the stress is linked to donor side related stress. If Area increases, the stess factor is an acceptor side linked stress.
  10. PIABS

    = Perfomance index on an absorption basis.
  11. Fv/Fm

    = Optimal quantum yield.

Derived Values in Measuring File

  1. Fv

    = Fm - Fo (maximal variable fluorescence)
  2. VJ

    = (Fj - Fo) / (Fm - Fo) (variable fluorescence for J step) represents the fraction of closed reaction centers at the J step.
  3. Fm / Fo

    = Fm / Fo
  4. Fv / Fo

    = Fm - Fo / Fo
  5. Fv / Fm

    = Tro /ABS or Fm - Fo / Fm. Optimal fluorescence yield.
  6. Mo

    = 4(F270us – F20us) / Fm-F20us) Approx initial slope in ms-1.
  7. Sm

    = area / Fm – F20us Multiple turn-over, or reductions of QA. Normalized Area used to compare different samples.
  8. Ss

    = Vj/Mo The smallest Sm turn-over (single turn-over or reduction of QA) representing the area corresponding to the O to J phase.
  9. N

    = Sm (Mo) (I / Vj) Turn-over number: How many timese QA has been reduced from 0 to tFM.
  10. Vav

    = 1 - (Sm/tFm) Average relative variable fluorecense from t=0 to tFm.