Continuous scanning micro-photolysis: A simple laser scanning microscopic method for lateral transport measurements employing single- or two-photon excitation

A comparatively simple laser scanning microscopic method for the determination of lateral diffusion coefficients at high temporal and spatial resolution is described. Combining two previously developed methods, continuous fluorescence microphotolysis and scanning microphotolysis, the new method is referred to as continuous scanning microphotolysis (continuous SCAMP). The principle of the method is simply to operate a commercial laser scanning microscope in the line scanning mode while monitoring the fluorescence emitted from the continuously scanned line as an x–t ‘image’. Fluorescence excitation can be effected by either single- or two-photon absorption. In the former case a standard, low power ion laser is sufficient while for two-photon excitation a femtosecond-pulsed titan sapphire laser can be employed. In both cases the laser beam power is adjusted such that a substantial but not excessive degree of photobleaching is induced. x–t images are evaluated so as to determine the dependence of the scanned line intensities on the scanning time. The fluorescence decay curves obtained in this manner are evaluated in terms of diffusion coefficients and photobleaching rate constants by numerical simulation of appropriate diffusion-reaction systems. The validity of the experimental and theoretical procedures was tested by measurements on a simple well-defined model system. The results suggested that the continuous SCAMP, when using single-photon excitation, is a particularly simple and sensitive method for determining lateral diffusion in two-dimensional systems such as cell membranes. Employing two-photon excitation, on the other hand, provides the continuous SCAMP with the capability for studying three-dimensional diffusion within cells, cell organelles and similar systems by still comparatively simple means. Keywords: confocal microscopy, continuous fluorescence microphotolysis, fluorescence photobleaching, lateral diffusion, two-photon excitation