Interferometric fringe fluorescence photobleaching recovery interrogates entire cell surfaces.
Interferometric fringe fluorescence photobleaching recovery interrogates entire cell surfaces.
复制标题
干涉条纹荧光光漂白恢复询问整个细胞表面。
DOI：
10.1016/s0006-3495(98)77602-7
复制
发表时间：
1998
影响因子：
3.4
通讯作者：
Barisas,BG
中科院分区：
文献类型：
--
作者：
Munnelly,HM;Roess,DA;Wade,WF;Barisas,BG
Fluorescence photobleaching recovery (FPR) measurements of cell surface protein lateral diffusion typically employ an interrogated spot of 0.5μm 1/e2radius. The effective spot area represents only 1/500 of the total surface of an 8-μm cell. An FPR measurement of a protein expressed as 50,000 copies per cell reflects the dynamics of 100molecules. This limits the precision and reproducibility of FPR measurements. We describe a method for interferometric fringe pattern FPR that permits simultaneous interrogation of the entire cell's surface. Fringe patterns are generated interferometrically within the optical path of an FPR system. Methods for interpreting fluorescence recovery kinetics on cells and for determining the protein mobile fraction are presented. With fringe FPR, the murine major histocompatibility complex class II antigen I-Akexpressed on M12.C3.F6 cells has 100-fold improved fluorescence signals relative to spot FPR, with corresponding improvements in signal-to-noise ratios of recovery traces. Diffusion coefficients (± standard deviation) of (2.1±0.4)×10−10and (1.8±1.0)×10−10cm2s−1with corresponding mobile fractions of I-Akof 66.1±7.8% and 63.4±18.0% were obtained by fringe and spot methods, respectively. The improved reproducibility of fringe over spot results is less than signal improvements predict. There may thus be substantial variation from cell to cell in protein dynamics, and this method may permit the assessment of such variation.
