title: Two-photon scanning microphotolysis for three-dimensional data storage and biological transport measurements.
abstract: 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 photoactivation. 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 approximately 40%. Under these conditions the spatial accuracy of photobleaching was 0.5 +/- 0.1 micron in the lateral (x.y) and 3.5 +/- 0.5 micron in the axial (z) direction, without deconvolution accounting for the optical resolution. Deconvolution improved the accuracy values by approximately 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.
authors.affiliation: Institut für Medizinische Physik und Biophysik, Westfälische Wilhelms-Universität, Münster, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Kubitscheck
authors.forename: M
authors.initials: M
authors.lastname: Tschödrich-Rotter
authors.forename: P
authors.initials: P
authors.lastname: Wedekind
authors.forename: R
authors.initials: R
authors.lastname: Peters
mesh_terms: D001427:Bacterial Toxins; D001692:Biological Transport; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007473:Ion Channels; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods*; D010782:Photolysis; D052899:Pore Forming Cytotoxic Proteins
chemical_list: D001427:Bacterial Toxins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007473:Ion Channels; D052899:Pore Forming Cytotoxic Proteins; C024098:6-carboxyfluorescein; C032676:aerolysin