High Probe Intensity Photobleaching Measurement of Lateral Diffusion in Cell Membranes |
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Authors: | Guy?M?Hagen Deborah?A?Roess Gildardo?Cruz?de?León Email author" target="_blank">B?George?BarisasEmail author |
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Institution: | (1) Department of Chemistry, Colorado State University, Fort Collins, Colorado, USA;(2) Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado, USA |
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Abstract: | Lateral diffusion measurements, most commonly accomplished through Fluorescence Photobleaching Recovery (FPR or FRAP), provide
important information on cell membrane molecules' size, environment and participation in intermolecular interactions. However,
serious difficulties arise when these techniques are applied to weakly expressed proteins of either of two types: fusions
of membrane receptors with visible fluorescent proteins or membrane molecules on autofluorescent cells. To achieve adequate
sensitivity in these cases, techniques such as interference fringe FPR are needed. However, in such measurements, cytoplasmic
species contribute to the fluorescence recovery signal and thus yield diffusion parameters not properly representing the small
number of surface molecules. A new method helps eliminate these difficulties. High Probe Intensity (HPI)-FPR measurements
retain the intrinsic confocality of spot measurements to eliminate interference from fluorescent cytoplasmic species. However,
HPI-FPR methods lift the previous requirement that FPR procedures be performed at probe beam intensities low enough to not
induce bleaching in samples during measurements. The high probe intensities now employed provide much larger fluorescence
signals and thus more information on molecular diffusion from each measurement. We report successful measurement of membrane
dynamics by this technique. |
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Keywords: | Photobleaching FPR FRAP membrane green fluorescent protein diffusion |
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