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Biophysical Journal, Volume 79, Issue 3, 1 September 2000, Pages 1655-1669
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Abstract | Full Text | PDF (542 kb) - Lateral Mobility of Membrane-Binding Proteins in Living Cell...Lateral Mobility of Membrane-Binding Proteins in Living Cells Measured by Total Internal Reflection Fluorescence Correlation Spectroscopy
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Abstract | Full Text | PDF (482 kb) - Lighting up the cell surface with evanescent wave microscopy...Lighting up the cell surface with evanescent wave microscopy
Trends in Cell Biology, Volume 11, Issue 7, 1 July 2001, Pages 298-303
Derek Toomre and Dietmar J. Manstein
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Abstract | Full Text | PDF (307 kb) - …more
Copyright © 1996 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 71, Issue 2, 1140-1151, 1 August 1996
doi:10.1016/S0006-3495(96)79316-5
Research Article
Cytoplasmic viscosity near the cell plasma membrane: translational diffusion of a small fluorescent solute measured by total internal reflection-fluorescence photobleaching recovery
R. Swaminathan, S. Bicknese, N. Periasamy and A.S. Verkman
Department of Medicine, University of California, San Francisco 94143–0521, USA.
Abstract
Total internal reflection-fluorescence recovery after photobleaching (TIR-FRAP) was applied to measure solute translational diffusion in the aqueous phase of membrane-adjacent cytoplasm. TIR fluorescence excitation in aqueous solutions and fluorescently labeled cells was produced by laser illumination at a subcritical angle utilizing a quartz prism; microsecond-resolution FRAP was accomplished by acousto-optic modulators and electronic photomultiplier gating. A mathematical model was developed to determine solute diffusion coefficient from the time course of photobleaching recovery, bleach time, bleach intensity, and evanescent field penetration depth; the model included irreversible and reversible photobleaching processes, with triplet state diffusion. The validity and accuracy of TIR-FRAP measurements were first examined in aqueous fluorophore solutions. Diffusion coefficients for fluorescein isothiocyanate-dextrans (10–2000 kDa) determined by TIR-FRAP (recovery t1/2 0.5–2.2 ms) agreed with values measured by conventional spot photobleaching. Model predictions for the dependence of recovery curve shape on solution viscosity, bleach time, and bleach depth were validated experimentally using aqueous fluorescein solutions. To study solute diffusion in cytosol, MDCK epithelial cells were fluorescently labeled with the small solute 2',7'-bis-2-carboxyethyl-5-carboxyfluorescein-acetoxymethyl-ester (BCECF). A reversible photobleaching process (t1/2 approximately 0.5 ms) was identified that involved triplet-state relaxation and could be eliminated by triplet-state quenching with 100% oxygen. TIR-FRAP t1/2 values for irreversible BCECF bleaching, representing BCECF translational diffusion in the evanescent field, were in the range 2.2–4.8 ms (0.2–1 ms bleach times), yielding a BCECF diffusion coefficient 6–10-fold less than that in water. These results establish the theory and the first experimental application of TIR-FRAP to measure aqueous-phase solute diffusion, and indicate slowed translational diffusion of a small solute in membrane-adjacent cytosol.
