INTERFEROMETRIC FRINGE FLUORESCENCE PHOTOBLEACHING RECOVERY INTERROGATES ENTIRE CELL-SURFACES

Fluorescence photobleaching recovery (FPR) measurements of cell surfac e protein lateral diffusion typically employ an interrogated spot of 0 .5 mu m 1/e(2) radius. The effective spot area represents only 1/500 o f the total surface of an 8-mu m cell, An FPR measurement of a protein expressed as 50,000 copies per cell reflects the dynamics of 100 mole cules. This limits the precision and reproducibility of FPR measuremen ts. We describe a method for interferometric fringe pattern FPR that p ermits 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 kinetic s on cells and for determining the protein mobile fraction are present ed, With fringe FPR, the murine major histocompatibility complex class II antigen I-Ak expressed on M12.C3.F6 cells has 100-fold improved fl uorescence signals relative to spot FPR, with corresponding improvemen ts in signal-to-noise ratios of recovery traces. Diffusion coefficient s (+/- standard deviation) of (2.1 +/- 0.4) x 10(-10) and (1,8 +/- 1.0 ) x 10(-10) cm(2) s-(1) with corresponding mobile fractions of I-A(k) of 66.1 +/- 7.8% and 63.4 +/- 18.0% were obtained by fringe and spot m ethods, respectively. The improved reproducibility of fringe over spot results is less than signal improvements predict. There may thus be s ubstantial variation from cell to cell in protein dynamics, and this m ethod may permit the assessment of such variation.