Interferometric fringe fluorescence photobleaching recovery interrogates entire cell surfaces.
Fluorescence photobleaching recovery (FPR) measurements of cell surface protein lateral diffusion typically employ an interrogated spot of 0.5 microm 1/e2 radius. The effective spot area represents only 1/500 of the total surface of an 8-microm cell. An FPR measurement of a protein expressed as 50,000 copies per cell reflects the dynamics of 100 molecules. 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-Ak expressed 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) x 10(-10) and (1.8 +/- 1.0) x 10(-10) cm2 s-1 with corresponding mobile fractions of I-Ak of 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.
Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523, USA. barisas@lamar.colostate.edu
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Barisas
D000818:Animals; D002308:Cardiolipins; D002460:Cell Line; D002784:Cholesterol; D004058:Diffusion; D004867:Equipment Design; D000949:Histocompatibility Antigens Class II / Q000096:biosynthesis / Q000737:chemistry*; D007700:Kinetics; D008081:Liposomes; D051379:Mice; D008857:Microscopy, Interference / Q000295:instrumentation / Q000379:methods; D008962:Models, Theoretical; D010713:Phosphatidylcholines; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods; D014162:Transfection
D002308:Cardiolipins; D000949:Histocompatibility Antigens Class II; D008081:Liposomes; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D002784:Cholesterol