Two-photon scanning microphotolysis for three-dimensional data storage and biological transport measurements

Scanning microphotolysis is a method that permits the user to select, within the scanning field of a confocal microscope, areas of arbitrary geometry for photobleaching or photo-activation, Two-photon absorption, by contrast, confers on laser scanning microscopy a true spatial selectivity by restricting excitation to very small focal volumes. In the present study the two methods were combined by complementing a laser scanning microscope with both a fast programmable optical switch and a titan sapphire laser, The efficiency and accuracy of fluorescence photobleaching induced by two-photon absorption were determined using fluorescein-containing polyacrylamide gels. At optimal conditions a single scan was sufficient to reduce the gel fluorescence by approximate to 40%. Under these conditions the spatial accuracy of photobleaching was 0.5 +/- 1 mu m in the lateral (x,y) and 3.5 +/- 0.5 mu m in the axial (z) direction, without deconvolution accounting for the optical resolution. Deconvolution improved the accuracy values by approximate to 30%. The method was applied to write complex three-dimensional patterns into thick gels by successively scanning many closely spaced layers, each according to an individual image mask. Membrane transport was studied in a model tissue consisting of human erythrocyte ghosts carrying Large transmembrane pores and packed into three-dimensional arrays, Upon equilibration with a fluorescent transport substrate single ghosts could be selectively photobleached and the influx of fresh transport substrate be monitored, The results suggest that two-photon scanning microphotolysis' provides new possibilities for the optical analysis and manipulation of both technical and biological microsystems.