A localized pattern photobleaching method for the concurrent analysis of rapid and slow diffusion processes.

A scanning pattern photobleaching method for the analysis of lateral transport is described and discussed. Fluorescence bleaching with a localized pattern allows for the concurrent analysis of motions over two very different characteristic distances: xi 0(-1), the repeat distance of the pattern, and W, the linear dimension of the illuminated region. The former motion is deduced from the decay of the modulation amplitude (of period xi 0(-1) of fluorescence scans with the attenuated pattern, the latter from the recovery of the average fluorescence intensity. Such analysis should prove useful for the study of samples with a wide range of diffusion coefficients, and for the separation of effects arising from lateral diffusion and association dynamics. Theoretical analyses are presented for three related problems: (a) the effect of pattern localization on the decay of the modulation amplitude, (b) the effect of the pattern modulation on the recovery of the average local fluorescence intensity, and (c) the effect of a limited diffusion space (with linear dimensions of only a few pattern periods) on the decay of the modulation amplitude.