Fluorescence pattern photobleaching recovery for samples with multi-component diffusion.

The translational mobility of proteins and lipids in phospholipid bilayers is often not well described as ideal self diffusion. One of the best methods for characterizing such non-ideal diffusion is to use fluorescence pattern photobleaching recovery. In this method, the spatial gradient of the monitoring and bleaching intensity is created by using epi-fluorescence and an expanded Gaussian-shaped laser beam which passes though a Ronchi ruling placed at the back image plane of a microscope. A difficulty arises when the fluorescence recovery from the exchange of slowly diffusing molecules between illuminated and non-illuminated stripes temporally overlaps with the recovery from the exchange of more rapidly diffusing molecules through the gradient produced by the broad Gaussian shape of the illumination. In the work presented here, a general theory is developed that describes the shape of the resulting fluorescence recovery curve for these typical experimental conditions. Approximate expressions amenable to non-linear curve fitting are also given. The new theoretical formalism has been demonstrated on data for the translational mobility of a fluorescent lipid probe in phospholipid bilayers deposited on planar-fused silica substrates.