Three‐dimensional diffusion measurements by scanning microphotolysis

Fluorescence microphotolysis techniques have been used widely to measure lateral diffusion in two‐dimensional microsystems such as cell membranes. However, a general microphotolysis method for the analysis of truly three‐dimensional diffusion processes has not been developed so far. Here we combine microphotolysis with confocal laser scanning microscopy and numerical data evaluation in such a manner that small volumes (≥ 0.5 μm3) can be photolysed within extended three‐dimensional samples and that fluorescence changes can be monitored at high time resolution (measuring interval 0.5 ms) and evaluated for lateral diffusion coefficients. We show furthermore that diffusion measurements can be performed according to three different experimental modes: (i) the instantaneous mode, (ii) the continuous mode and (iii) the mixed mode. For the evaluation of experimental data in terms of diffusion coefficients we have developed and thoroughly characterized a theoretical framework which is based on the numerical simulation of appropriate reaction–diffusion systems. The theoretical framework is rather general and flexible and can be applied to any microphotolysis geometry, makes provision for photolysis during fluorescence monitoring and takes into account the convolution of concentration distributions with imaging point spread functions. The new method was tested employing simple well‐defined model systems.