A microscope for two-dimensional measurements of in vivo chlorophyll fluorescence kinetics using pulsed measuring radiation, continuous actinic radiation, and saturating flashes

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 mic ro-channel plate image amplifier. The microscopic field is irradiated simul taneously by three types of radiation: actinic radiation, saturating flashe s, and pulsed measuring radiation. The measuring pulses are generated by a light-emitting diode and their duration is between 10 to 250 mus The detect ion of fluorescence images (300x400 pixels, 8 bit) has a maximum time resol ution of 40 ms and is gated in synchrony with the exciting pulses. This all ows measuring on a background of a continuous actinic radiation up to irrad iance that can elicit the maximal fluorescence yield (F-M). On the other ha nd, the integral irradiance of the objects by the measuring radiation is ve ry low, e.g., 0.08 mu mol m(-2) s(-1) at 0.5 mum spatial resolution and 0.0 06 mu mol m(-2) s(-1) at 4 mum spatial resolution. This allows a reliable r ecording of F-0 even in very short time intervals (e.g., 5x80 ms). The soft ware yields fluorescence kinetic curves for objects in user-selected areas as well as complete false-colour maps of the essential fluorescence kinetic s parameters (F-M, F-0, F-V, F-V/F-M, etc.) showing a two-dimensional distr ibution of their values. Several examples demonstrate that records of fluor escence kinetics can be obtained with a reasonable signal-to-noise ratio wi th all standard microscope objectives and with object sizes reaching from s egments of leaf tissue to individual algal cells or chloroplasts.