Combined Gas Exchange
and Fluorescence

diagram of gas exchange

Gas exchange is useful to characterize leaf-level CO2 and H2O flux, stomatal conductance, and more, but this is only part of the story.

diagram of chlorophyll fluorescence

Pulsed amplitude modulation (PAM) chlorophyll a fluorescence measurements provide information about the light-driven electron transfer rate (ETR), non-photochemical quenching (NPQ), and an assortment of reactions that collectively protect a leaf when it absorbs excessive light energy.

Photosynthesis measurements, when made with gas exchange and fluorescence together, can provide a more complete understanding of photosynthetic processes.

What can you learn from combined gas exchange and fluorescence measurements?

Integrated analyses of gas exchange and chlorophyll a fluorescence over the same leaf area can provide a more complete picture of photosynthesis than when using either technique alone. For example, when used together, these techniques can provide one way to assess the pathway of CO2 diffusion from the intercellular leaf air space to the chloroplast.

mesophyll resistance

The resistance of CO2 along this pathway is currently the subject of intense research aimed at improving water use efficiency in plants. Given that water stress limits plant productivity worldwide, understanding these diffusive processes is highly important.

How does the LI-6800 Multiphase Flash Fluorometer improve combined gas exchange and fluorescence measurements?

Uniform lighting of the leaf surface

Understanding leaf-level processes from an integrated assessment of gas exchange and fluorescence requires that the leaf area under investigation be illuminated by a highly uniform light source. If not, gas exchange parameters can be artifactually inaccurate, resulting in errors that propagate to many other parameters.

The LI-6800 fluorometer light source is highly uniform (i.e. light intensities of ±10% of the mean over 90% of the leaf area). This high light uniformity minimizes artifacts that can cause errors in gas exchange measurements and confound the understanding of processes that require comparison of gas exchange and PAM chlorophyll a fluorescence parameters.

other fluorometers the LI-6800 fluorometer

High-intensity flashes

The maximum fluorescence yield must be known in order to accurately estimate ETR and NPQ, as well as several other important parameters. Historically, estimation of the maximum fluorescence yield has been achieved using short (one second or less), high intensities (many times higher than full sunlight) flashes of light termed ‘saturation flashes’. The yield of fluorescence increases hyperbolically towards an asymptote in response to progressively increasing light intensity, at which the maximum fluorescence yield can be most accurately approximated.

Estimation of the maximum fluorescence yield, called Fm’, when measured in an illuminated leaf, requires the use of extreme light intensities never encountered by plants in nature. Furthermore, estimation of Fm’ requires even higher intensities as NPQ increasingly develops in a leaf, which begins to occur at light intensities well below full sunlight. The LI-6800 fluorometer can deliver traditional saturation flashes upwards of 16,000 µmol m-2 s-1 over a leaf area of 6 cm2; something no other commercial fluorometer is capable of doing.

Multiphase Flash™ Fluorescence to accurately estimate Fm’

chart of fm prime
Accurate measurements of Fm’ through to other calculated fluorescence parameters, ensuring accurate determination of non-photochemical quenching (NPQ), energy dependent quenching (qE), the quantum yield of photosystem II-mediated electron transport (ΦPSII), the actual measured electron transport rate (J), and mesophyll conductance (gm).

An innovative technique known as Multiphase Flash Fluorescence can be used to quickly estimate, using a single flash event, the fluorescence yield at infinite irradiance, a more accurate determination of “true” Fm’. This value, known as extrapolated maximum fluorescence yield (EFm’), is a more accurate assessment of “true” Fm’, contrary to traditional measurements of apparent maximum fluorescence yield (AFm’) using traditional rectangular flashes.

Experimental research shows that EFm’ values are invariably higher than AFm’ values measured using increasingly intense saturating flashes of light (Q’). In some instances, erroneous estimates of Fm’ can subsequently result in errors in estimation of ΦPSII by as much as 15-30%. Furthermore, while the measured values of AFm’ progressively increase with increasing Q’, estimates of EFm’ measured across the same range of Q’ intensities are constant, except at the lowest Q’, indicating that accurate estimates of Fm’ can be achieved at lower flash intensities using the Multiphase Flash Fluorescence method. As such, since this technique can be used to estimate Fm’ using moderate flash intensities, plants that are sensitive to photodamage can be studied with less risk of damage from the saturation flash.

Download stressed plants document

EFm’ from the LI-COR Multiphase Flash(TM) Fuorescence
Estimates of EFm’ and AFm’ as a function of saturation pulse irradiance (Q’) in chamber-grown Nicotiana tobacum. Rectangular flashes tend to underestimate true Fm’.

Optimized signal-to-noise ratio

An aspect of obtaining accurate fluorescence parameters concerns the signal-to-noise ratio. This is especially important during fluorescence induction curves, when data points are being acquired very rapidly and undergoing minimal averaging.

The LI-6800 uses a carefully refined protocol, in which modulated light is altered solely by changing the frequency of the modulated pulses, not the pulse amplitude or the pulse width. This ultimately prevents the induction of photosynthesis by the measuring light in both dark- and light-adapted leaves and optimizes the fluorescence signal. The LI-6800 provides predetermined setpoints that optimize the signal-to-noise ratio without violating the PAM chlorophyll a fluorescence technique.

High frequency fluorometer for induction kinetics

Capable of modulating the measuring light at frequencies up to 250 kHz (250,000 samples per second, in contrast with 20 kHz available with the LI-6400XT), the LI-6800 fluorometer can fully characterize the fluorescence induction transient of a leaf with high resolution.

OJIP curve

Fluorescence induction (OJIP) curve

Fluorescence induction curves serve as a means of rapidly assessing exquisite photophysical details of a key pigment-binding protein complex, PSII, in which photosynthesis is initiated. The different transitions of an induction curve (historically termed “OJIP transient”) can be used to measure environmental stress in plants. The LI-6800 fluorometer is unique in that it is capable of measuring two types of fluorescence during induction: 1) modulated; and 2) continuous. The photophysical reactions underlying an OJIP transient occur over a range of time scales. These time scales of the OJIP transient require that measurements be capable of occurring on time scales ranging from microseconds to milliseconds. The LI-6800 fluorometer can acquire modulated and continuous fluorescence signals on time scales ranging from 4 microseconds to hundreds of milliseconds.

Measurements & Applications

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