Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy

N. L. Thompson, Thomas P Burghardt, D. Axelrod

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Abstract

The theoretical basis of a new technique for measuring equilibrium adsorption/desorption kinetics and surface diffusion of fluorescent-labeled solute molecules at solid surfaces has been developed. The technique combines total internal reflection fluorescence (TIR) with either fluorescence photobleaching recovery (FPR) or fluorescence correlation spectroscopy (FCS). A laser beam totally internally reflects at a solid/liquid interface; the shallow evanescent field in the liquid excites the fluorescence of surface adsorbed molecules. In TIR/FPR, adsorbed molecules are bleached by a flash of the focused laser beam; subsequent fluorescence recovery is monitored as bleached molecules exchange with unbleached ones from the solution or surrounding nonilluminated regions of the surface. In TIR/FCS, spontaneous fluorescence fluctuations due to individual molecules entering and leaving a well-defined portion of the evanescent field are autocorrelated. Under appropriate experimental conditions, the rate constants and surface diffusion coefficient can be readily obtained from the TIR/FPR and TIR/FCS curves. In general, the shape of the theoretical TIR/FPR and TIR/FCS curves depends in a complex manner upon the bulk and surface diffusion coefficients, the size of the illuminated or observed region, and the adsorption/desorption kinetic rate constants. The theory can be applied both to specific binding between immobilized receptors and soluble ligands, and to nonspecific adsorption processes. A discussion of experimental considerations and the application of this technique to the adsorption of serum proteins on quartz may be found in the accompanying paper. (Burghardt and Axelrod. 1981. Biophys. J. 33:455).

Original languageEnglish (US)
Pages (from-to)435-454
Number of pages20
JournalBiophysical Journal
Volume33
Issue number3
StatePublished - 1981
Externally publishedYes

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Fluorescence Recovery After Photobleaching
Spectrum Analysis
Fluorescence
Fluorescence Spectrometry
Adsorption
Lasers
Quartz
Blood Proteins

ASJC Scopus subject areas

  • Biophysics

Cite this

Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy. / Thompson, N. L.; Burghardt, Thomas P; Axelrod, D.

In: Biophysical Journal, Vol. 33, No. 3, 1981, p. 435-454.

Research output: Contribution to journalArticle

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