Photosynthetica 2000, 38(4):553-570 | DOI: 10.1023/A:1012461407557

A Microscope for Two-Dimensional Measurements of In Vivo Chlorophyll Fluorescence Kinetics Using Pulsed Measuring Radiation, Continuous Actinic Radiation, and Saturating Flashes

H. Küpper1,2,3,4, I. Šetlík2,3,5, M. Trtílek5, L. Nedbal6
1 Faculty of Sciences, Department of Biology, University of Konstanz, Konstanz, Germany
2 Microbiological Institute, Academy of Sciences of the Czech Republic, Czech Republic
3 Department of Autotrophic Microorganisms, Opatovický mlýn, Třeboň, Czech Republic
4 Biological Faculty, University of South Bohemia, České Budějovice, Czech Republic
5 Biological Faculty, University of South Bohemia, České Budějovice, Czech Republic
6 Institute of Landscape Ecology, Academy of Sciences of the Czech Republic, Photosynthesis Research Centre, Zámek, Nové Hrady, Czech Republic

Transients of chlorophyll fluorescence in photosynthetic objects are often measured using short pulses of exciting radiation, which has recently been employed to capture kinetic images of fluorescence at the macroscopic level. Here we describe an instrument introducing this principle to recording of two dimensional fluorescence transients in microscopic objects. A modified fluorescence microscope is equipped with a CCD camera intensified by a micro-channel plate image amplifier. The microscopic field is irradiated simultaneously by three types of radiation: actinic radiation, saturating flashes, and pulsed measuring radiation. The measuring pulses are generated by a light-emitting diode and their duration is between 10 to 250 µs. The detection of fluorescence images (300×400 pixels, 8 bit) has a maximum time resolution of 40 ms and is gated in synchrony with the exciting pulses. This allows measuring on a background of a continuous actinic radiation up to irradiance that can elicit the maximal fluorescence yield (FM). On the other hand, the integral irradiance of the objects by the measuring radiation is very low, e.g., 0.08 µmol m-2 s-1 at 0.05 µm spatial resolution and 0.006 µmol m-2 s-1 at 4 µm spatial resolution. This allows a reliable recording of F0 even in very short time intervals (e.g., 5×80 ms). The software yields fluorescence kinetic curves for objects in user-selected areas as well as complete false-colour maps of the essential fluorescence kinetics parameters (FM, FO, FV, FV/FM, etc.) showing a two-dimensional distribution of their values. Several examples demonstrate that records of fluorescence kinetics can be obtained with a reasonable signal-to-noise ratio with all standard microscope objectives and with object sizes reaching from segments of leaf tissue to individual algal cells or chloroplasts.

Prepublished online: August 1, 2000; Published: November 1, 2000  Show citation

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Küpper, H., Šetlík, I., Trtílek, M., & Nedbal, L. (2000). A Microscope for Two-Dimensional Measurements of In Vivo Chlorophyll Fluorescence Kinetics Using Pulsed Measuring Radiation, Continuous Actinic Radiation, and Saturating Flashes. Photosynthetica, 38(4), 553-570. doi: 10.1023/A:1012461407557
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