Continuous fluorescence microphotolysis: A sensitive method for study of diffusion processes in single cells

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Continuous fluorescence microphotolysis is a sensitive method for the study of translational diffusion in the plasma membrane of single living cells and related systems. In this communication the conceptual basis of the method and its theoretical framework and experimental realization, as well as applications, are reported. In continuous fluorescence microphotolysis a microscopic membrane area of a single fluorescently labeled cell is irradiated by a laser beam while the fluorescence emitted from the area is monitored. The decay of the measuring signal reflects the competition of two processes: (i) the elimination of fluorophores by irreversible photolysis, and (ii) the entrance of new fluorophores into the area by diffusion. Rate constants for the two processes can be derived from the measuring data by mathematical analysis. As compared to our initial approach, fluorescence microphotolysis [Peters, R., Peters, J., Tews, K. H. & Bähr, W. (1974) Biochim. Biophys. Acta 367, 282-294], the main advantage of the method described here is an improvement of data quality and detection limit by orders of magnitude. From the practical point of view the main advantage is a simplification of the experimental setup. Results obtained by this method are encouraging and support the contention that continuous fluorescence microphotolysis may disclose new aspects of diffusion processes in biological systems.

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