Integrated Fluorescence Measurements with the LI-6400XT

The Leaf Chamber Fluorometer is a pulse-amplitude modulated (PAM) fluorometer that can be used to take measurements on both dark- and light-adapted samples. Measured parameters include Fo, Fm, F, Fm', and Fo', and calculated parameters include Fv, Fv/Fm, dF/Fm, qP, qN, NPQ, and ETR.

The 6400-40 provides complete control over the actinic and saturation (independently controlled red 630 nm and blue 470 nm LEDs), measuring (red 630 nm LEDs, modulated from 0.25 to 20 kHz), and far-red (740 nm LED for PSI excita-tion) light.

The unique design of the 6400-40 Leaf Chamber Fluorometer eliminates the need for fragile, awkward fiber optic light guides. Lightweight design and low power consumption make it possible for one person to gather data quickly and easily. Calibration information for the Leaf Chamber Fluorometer is stored onboard, making it easy to move between different LI-6400XT consoles.

Assessing Fv in either light- or dark-adapted measurements requires application of a saturating flash of light to fully reduce QA, the primary acceptor of PSII. In many conditions, especially high-light adapted field plants, it is difficult to achieve full reduction of the QA-PQ pool with the usual rectangular flash (RF, Figure 1) method, which results in an underestimation of Fm', ?PSII, and ETR (Earl and Ennahli, 2004; Markgraf and Berry, 1990). The multiphase flash protocol (MPF, Figure 1) can be used under conditions when it is difficult to fully-saturate the plant material with a normal, rectangular flash (RF) in order to derive true estimates of Fm', ?PSII , and ETR within approximately one second (Loriaux et al., 2006).

With Version 6.0 OPEN software and above, the LI-6400/6400-40 System supports both the rectangular and multiphase types of saturating flashes. The RF type provides a single, saturating pulse of light for a short duration and Fm’ is determined from the maximum value of fluorescence during that pulse. The MPF type uses three phases, including a ramp of intensities, within a single flash of 1.5 to 2 s in length (Figure 1). Flash intensities and length are user-defined. After the multiphase flash is applied, Fm’ is determined from the y-intercept of a plot of fluorescence vs. 1/ intensity (Figure 2).

Figure 1. Rectangular flash (RF) method: a saturating multiturnover flash (Q) of 400 to 1200 ms duration. Multiphase flash (MPF) method: (1) high, nearly saturating Q for approximately 250 ms to reduce QA-PQ pool; (2) ramp of declining Q for about 500 ms; (3) return to the initial high Q for approximately 250 ms to check for flash-induced non-photochemical quenching (qN).

Figure 2. Fm' values from phase 2 of the MPF method are regressed against 1E4/Q and extrapolated to estimate the maximal fluorescence at infinite flash intensity.

References
Markgraf, T. and J. Berry. 1990. Measurement of photochemical and non-photochemical quenching: correction for turnover of PS2 during steady-state photosynthesis. Curr. Res. Photosynth. 4:279-282.
Earl, H.J. and S. Ennahli. 2004. Estimating photosynthesis electron transport via chlorophyll fluorometry without photosystem II light saturation. Photosynth. Res. 82:177-186.
Loriaux, S. D., R. A. Burns, J. M. Welles, D. K. McDermitt, and B. Genty. 2006. Determination of maximal chlorophyll fluorescence using a multiphase single flash of sub-saturating intensity. Poster Presentation. August, 2006. American Society of Plant Biologists Annual Meetings, Boston, MA.

An excellent article that references the use of the 6400-40 Leaf Chamber Fluorometer appeared in the December 2002 issue of Plant Physiology. The article, entitled "Temperature Response of Mesophyll Conductance. Implications for the Determination of Rubisco Enzyme Kinetics and for Limitations to Photosynthesis in Vivo" was written by Carl J. Bernacchi, Archie R. Portis, Hiromi Nakano, Susanne von Caemmerer, and Stephen P. Long.

Download a copy here.

A more recent reference of the 6400-40 Leaf Chamber Fluorometer can be found in the Journal of the American Society for Horticultural Science: S. Kumudini, 2004. Effect of Radiation and Temperature on Cranberry Photosynthesis and Characterization of Diurnal Change in Photosynthesis. J. Amer. Soc. Hort. Sci., vol. 129, no. 1, p. 106-111

You can read the abstract of the paper or the entire paper if you have an online subscription to the journal.