期刊
METHODS
卷 29, 期 2, 页码 142-152出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/S1046-2023(02)00305-5
关键词
single molecule; total internal reflection fluorescence microscopy; TIRF; GFP imaging; live-cell imaging
资金
- Medical Research Council [MC_U117570592] Funding Source: Medline
- MRC [MC_U117570592] Funding Source: UKRI
- Medical Research Council [MC_U117570592] Funding Source: researchfish
Over the past 10 years, advances in laser and detector technologies have enabled single fluorophores to be visualized in aqueous solution. Here, we describe methods based on total internal reflection fluorescence microscopy (TIRFM) that we have developed to study the behavior of individual protein molecules within living mammalian cells. We have used cultured myoblasts that were transiently transfected with DNA plasmids encoding a target protein fused to green fluorescent protein (GFP). Expression levels were quantified from confocal images of control dilutions of GFP and cells with 1-100 nM GFP were then examined using TIRFM. An evanescent field was produced by a totally internally reflected, argon ion laser beam that illuminated a shallow region (50100 nm deep) at the glass-water interface. Individual GFP-tagged proteins that entered the evanescent field appeared as individual, diffraction-limited spots of light, which were clearly resolved from background fluorescence. Molecules that bound to the basal cell membrane remained fixed in position for many seconds, whereas those diffusing freely in the cytoplasm disappeared within a few milliseconds. We developed automated detection and tracking methods to recognize and characterize the behavior of single molecules in recorded video sequences. This enabled us to measure the kinetics of photobleaching and lateral diffusion of membrane-bound molecules. (C) 2003 Elsevier Science (USA). All rights reserved.
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