Single-cell flux measurement by continuous fluorescence microphotolysis

M. Scholz, K. Schulten, R. Peters

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)37-44
Number of pages8
JournalEuropean Biophysics Journal
Volume13
Issue number1
DOIs
StatePublished - Oct 1 1985

Keywords

  • anion transport
  • erythrocyte membrane
  • Fluorescence microphotolysis
  • flux measurement
  • photobleaching

ASJC Scopus subject areas

  • Biophysics

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