Scanning microphotolysis: Three-dimensional diffusion measurement and optical single-transporter recording

Scanning microphotolysis (SCAMP) is a combination of fluorescence microphot olysis and confocal laser scanning microscopy. A laser scanning microscope is equipped with an optical switch able to modulate the power or/and wavele ngth of the laser beam in less than a microsecond while a dedicated compute r program is employed to precisely coordinate scanning process and laser be am modulation. By these means it becomes possible to vary the power or/and wavelength of the laser beam during scanning at a precision of one resoluti on element. Patterns of almost arbitrary design can be written into the obj ect by photolysis, e.g., photobleaching or photoactivation. The dissipation of the photolysis pattern by diffusion or other types of molecular transpo rt can be followed at confocal resolution and used to characterize the tran sport process. SCAMP can be employed in conjunction with single-photon or m ultiphoton excitation. Furthermore, it can be easily installed on virtually any confocal laser scanning microscope. We summarize at first the conceptu al and practical basis of SCAMP. Then, two novel applications are discussed : (i) measurements of translational diffusion coefficients in truly three-d imensional systems at diffraction-limited resolution, and (ii) optical reco rding of single transporters in membrane patches. (C) 1999 Academic Press.