Chlorophyll fluorescence at 77 K in intact leaves: Characterization of a technique to eliminate artifacts related to self-absorption.

Due to the optical density of photosynthetic tissues the spectral characteristics of fluorescence emitted at 77 K directly from frozen plant material are distorted by differential re-absorption of the emitted light: the emission band related to PSII can be lowered by more than 80%, relative to the PSI band and the profile of the excitation spectra becomes flattened. It is demonstrated that such distortion cannot be neglected as its extent varies from sample to sample. A technique is introduced to eliminate sample artifacts related to self-absorption: subcellular small particles are prepared from rapidly cooled leaves and then 'diluted' without re-thawing at a concentration corresponding to about 5 μg chlorophyll·cm(-3) into a matrix consisting of ice and quartz particles. The photochemical pigment apparatus is expected to remain fixed in the in vivo state. Different kinds of plant material is used and it is demonstrated how this preparative approach allows to study the in vivo distribution of energy between the two photosystems from pure 77 K spectrofluorimetry, even when the optical properties of whole leaves or thalli normally would exclude quantitative analysis.