Scanning microphotolysis: A new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging

Abstract

Summary The fluorescence photobleaching method has been widely used to study molecular transport in single living cells and other microsystems while confocal microscopy has opened new avenues to high‐resolution, three‐dimensional imaging. A new technique, scanning microphotolysis (Scamp), combines the potential of photobleaching, beam scanning and confocal imaging. A confocal scanning laser microscope was equipped with a sufficiently powerful laser and a novel device, the ‘Scamper’. This consisted essentially of a filter changer, an acousto‐optical modulator (AOM) and a computer. The computer was programmed to activate the AOM during scanning according to a freely defined image mask. As a result almost any desired pattern could be bleached (‘written’) into fluorescent samples at high definition and then imaged (‘read’) at non‐bleaching conditions, employing full confocal resolution. Furthermore, molecular transport could be followed by imaging the dissipation of bleach patterns. Experiments with living cells concerning dynamic processes in cytoskeletal filaments and the lateral mobility of membrane lipids suggest a wide range of potential biological applications. Thus, Scamp offers new possibilities for the optical manipulation and analysis of both technical and biological microsystems.

An optical method for recording the activity of single transporters in membrane patches

“…Optical single-transporter recording was largely improved by the recent development of scanning microphotolysis (SCAMP; Kubitscheck et al, 1994;Wedekind et al, 1994), a method combining fluorescence microphotolysis with laser scanning and confocal imaging. For scanning microphotolysis a commercial confocal laser scanning microscope is equipped with a fast optical switch allowing one to modulate the laser beam power during scanning according to a deliberately constructed image mask.…”

The biochemistry of RNA metabolism studied in situ

“…The FRAP experimental design was later adapted to three dimensional molecule mobility and laser scanning confocal microscopy. 18 The broadening of FRAP theory to include binding of fluorescent molecules to intracellular structures then allowed the measurement of binding constants. [13][14][15][19][20][21][22] The first uses of FRAP to study the dynamic function of nuclear proteins important in RNA metabolism were by the Misteli and Hendzell laboratories.…”

Heterogeneous photobleaching in confocal microscopy caused by differences in refractive index and excitation mode

“…This leads to the conclusion that photobleaching in confocal scanning laser microscopy is dependent on photon energy flux density (joule/m 2 s). Cytometry 32:137-146, 1998.1998 Wiley-Liss, Inc.Key terms: photobleaching; fading; confocal microscopy; fluorescence microscopy On the one hand, photobleaching and related photochemical processes are recognized experimental barriers to quantification in fluorescence microscopy (4,9,10,19,17,25), whereas, on the other hand, timeintegrated fluorescence microscopy can give valuable information about the physical environment of the molecule and can elucidate the dynamics of various cellular processes or can derive the energy transfer efficiency (1,3,5,13,16,27,38).Confocal microscopes are used to obtain three-dimensional images by piling up two-dimensional images from consecutive focal planes. During image scanning, the whole depth of the object is irradiated by a conical excitation light beam, but only light at the focal plane is recorded.…”

“…In the discontinuous mode (5,22,38), two greatly differing excitation intensities of laser light power are employed. Initially, fluorescence is measured at a low, nonphotolyzing beam power.…”