Single-cell flux measurement by continuous fluorescence microphotolysis |
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Authors: | M Scholz K Schulten R Peters |
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Institution: | (1) Max-Planck-Institut für Biophysik, Kennedyallee 70, D-6000 Frankfurt/Main 70, Federal Republic of Germany;(2) Physik-Department, Technische Universität München, Theoretische Physik, D-8046 Garching, Federal Republic of Germany |
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Abstract: | Continuous fluorescence microphotolysis (CFM) was adapted to flux measurements in single cells. The principle of the method is simple: Cells are equilibrated with a fluorescent solute, an individual cell is continuously irradiated by a laser beam focussed down to approximately the diameter of the cell, and fluorescence originating from the irradiated cell is monitored. In this procedure irradiation irreversibly photolyzes chromophores in the cell while fresh chromophores enter the cell by membrane transport (flux). The resulting fluorescence decay can be analyzed for the rate constants of both membrane transport and photolysis. As an experimental test of the new method the band-3 mediated transport of the fluorescent anion N-(7-nitrobenzofuranzan-4-yl)-taurine (NBD-taurine) across the erythrocyte membrane was measured. For various experimental conditions good agreement between values obtained by CFM and by fluorescence microphotolysis (FM) was observed. By measurements on single ghosts it was furthermore found that photolysis of NBD-taurine is first-order with respect to the power of irradiation. On this basis a stepped-intensity procedure was worked out that facilitates data evaluation in flux measurements. Also, by analysing the relations between CFM and FM flux measurements a method was devised by which FM data can be corrected for (inevitable) photolysis. |
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Keywords: | Fluorescence microphotolysis photobleaching flux measurement erythrocyte membrane anion transport |
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