Mobility measurement by analysis of fluorescence photobleaching recovery kinetics

D. Axelrod, D. E. Koppel, J. Schlessinger, E. Elson, W. W. Webb

Research output: Contribution to journalArticle

1865 Scopus citations

Abstract

Fluorescence photobleaching recovery (FPR) denotes a method for measuring two-dimensional lateral mobility of fluorescent particles, for example, the motion of fluorescently labeled molecules in approximately 10 mum2 regions of a single cell surface. A small spot on the fluorescent surface is photobleached by a brief exposure to an intense focused laser beam, and the subsequent recovery of the fluorescence is monitored by the same, but attenuated, laser beam. Recovery occurs by replenishment of intact fluorophore in the bleached spot by lateral transport from the surrounding surface. We present the theoretical basis and some practical guidelines for simple, rigorous analysis of FPR experiments. Information obtainable from FPR experiments includes: (a) identification of transport process type, i.e. the admixture of random diffusion and uniform directed flow; (b) determination of the absolute mobility coefficient, i.e. the diffusion constant and/or flow velocity; and (c) the fraction of total fluorophore which is mobile. To illustrate the experimental method and to verify the theory for diffusion, we describe some model experiments on aqueous solutions of rhodamine 6G.

Original languageEnglish
Pages (from-to)1055-1069
Number of pages15
JournalBiophysical Journal
Volume16
Issue number9
DOIs
StatePublished - Jan 1 1976
Externally publishedYes

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