Lateral diffusion measurements on genetically introduced fluorescent proteins

B. George Barisas; Deborah A. Roess; Gildardo Cruz de Leon; Guy M. Hagen

doi:10.1117/12.528772

14 June 2004 Lateral diffusion measurements on genetically introduced fluorescent proteins

B. George Barisas, Deborah A. Roess, Gildardo Cruz de Leon, Guy M. Hagen

Proceedings Volume 5329, Genetically Engineered and Optical Probes for Biomedical Applications II; (2004) https://doi.org/10.1117/12.528772
Event: Biomedical Optics 2004, 2004, San Jose, CA, United States

Abstract

Genetic introduction of fluorescent labels such as the Visible Fluorescent Proteins (VFP) has revolutionized the visualization and characterization of cellular proteins. Lateral diffusion measurements, most commonly accomplished through Fluorescence Photobleaching Recovery (FPR or FRAP), provide important information on such molecules’ size, environment and participation in intermolecular interactions. However, serious difficulties arise when these techniques are applied to VFP fusion proteins since cytoplasmic species contribute to the fluorescence recovery signal and thus distort measurements aimed at surface molecules. Two new methods help eliminate these difficulties through distinctly different strategies. In Total Internal Reflection Interference Fringe FPR, interfering laser beams enter a 1.65 NA Olympus objective at the periphery of the back focal plane where the NA exceeds 1.38. This creates an interference pattern totally internally reflected at the coverslip-medium interface. Fluorescence excitation occurs only where the cell contacts the coverslip so no contribution arises from cytoplasmic species. Alternatively, 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 measurements of membrane dynamics of various VFP species obtained by these techniques and compare them with results of earlier FPR methods which previously proved unsatisfactory in these instances.

Citation Download Citation

B. George Barisas, Deborah A. Roess, Gildardo Cruz de Leon, and Guy M. Hagen "Lateral diffusion measurements on genetically introduced fluorescent proteins", Proc. SPIE 5329, Genetically Engineered and Optical Probes for Biomedical Applications II, (14 June 2004); https://doi.org/10.1117/12.528772

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