Interferometric Fringe Patterns Interrogate Entire Cell Surfaces in Fluorescence Photobleaching Recovery Measurements of Lateral Diffusion

摘要

Lateral diffusion of cell surface proteins is commonly measured by spot fluorescence photobleaching recovery (FPR) methods where the 1/e{sup}2 radius of the interrogated spot is typically 0.5μm. On an 8μm lymphocyte the effective spot area represents only 1/500 of the total surface. An FPR lateral diffusion measurement of a protein expressed as 50,000 copies per cell thus reflects the dynamics of only 100 molecules and this greatly limits the precision and reproducibility of FPR measurements. A new method for interferometric fringe pattern FPR permits simultaneous interrogation of the entire surface of round cells. Fringe patterns are generated interferometrically within the optical path of a conventional microscope FPR system so that spot photobleaching measurements can be performed interchangeably. Methods for interpreting recovery kinetics on round cells and for determining the fraction of mobile protein are presented. Fringe FPR data of the murine MHC Class II antigen I-A{sup}k (wt) expressed on M12.C3.F6 cells showed fluorescence signals improved 100-fold relative to spot FPR, with corresponding improvements in S/N ratios of recovery traces. Diffusion coefficients of 2.07±0.37 and 1.79±0.97×10{sup}(-10) cm{sup}2 sec{sup}(-1) were obtained by fringe and spot methods, respectively. The corresponding mobile fractions of I-A{sup}k were 66.1±7.8% and 63.4±18.0%. Improved reproducibility of fringe over spot results are slightly 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. Supported in part by NIH grant AI36306 to BGB.