Abstract

Confocal microscopes improve resolution by creating optical sections with spatial filtering. These optical sections can be reconstructed to produce complete three-dimensional images suitable for studying a variety of important biological problems. However, over the time course of an image scan, confocal microscopes illuminate the full specimen volume with the excitation wavelength (as in standard fluorescence microscopy) which can limit the image quality. The use of a short optical pulse (from an ultra-short pulse laser) allows one to utilize two-photon excitation, which occurs only at the focus of the (high NA) objective, with several advantages following. Background scattered light is lower, and fluorophore bleaching only occurs at the objective focus instead of throughout the sample volume. Consequently, three-dimensional imaging experiments may be performed at higher excitation powers, greatly improving signal-to-noise, or lower excitation power could be used, and the number of image sets collected over time could be increased. Finally, since the excitation volume is lower with two-photon excitation, caged compounds may be released in a small (diffraction-limited) volume in a specimen. Thus, a new tool may be developed for the researcher a miniature (micron-cubed) opto-chemical probe that can be used in a very non-invasive way.

© 1994 Optical Society of America