Fluorescence confocal laser scanning microscopy for three-dimensional imaging of living biological specimens

These calculations and experiments are designed to advance an understanding of the confocal laser scanning microscope (CLSM) and to develop new CLSM capabilities for biological imaging. The CLSM combines tightly focused illumination and spatially filtered detection to reduce out-of-focus background and to image thin optical sections within thick, translucent specimens. The effect of the spatial filter (detector aperture) on background rejection is measured by the signal-to-background ratio (S/B). Signal (S) is defined as the fluorescence generated within a resolution volume determined by the size of the focused laser illumination, and background (B) is defined as the fluorescence originating outside the same volume. Both calculations and experiments show that S/B in the confocal microscope can be over 100 times greater than S/B in the conventional fullfield microscope. Values of S/B are also calculated for the following pseudoconfocal microscopes: spinning disk, line illumination, and slit detection. Calculations and experiments reveal that the shot noise limited signal-to-noise ratio (S/N) can be optimized by a detector aperture that rejects background without excessive signal loss. The optimal confocal S/N can be a factor of 10 greater than S/N in the fullfield microscope; optimal detector aperture sizes are calculated for each pseudoconfocal geometry.; Two new imaging modes of the laser scanning microscope that complement CLSM are presented. An instrument capable of simultaneous fullfield laser scanning microscopy (FLSM) and CLSM is described, and experiments are performed to compare their 3-d imaging properties. The conditions for equivalent imaging in the FLSM and conventional fullfield microscope are extended to three-dimensions, and the CLSM and fullfield microscope imaging properties are compared by transitive equality. Simultaneous differential interference contrast (DIC) and CLSM is also presented. This technique combines the axial resolution inherent in both DIC and CLSM to allow three-dimensionally resolved structure-composition correlations.; Simultaneous DIC-CLSM is used to measure the correlated motions of crosslinked IgE receptors and membrane ruffles on the surface of RBL cells. Direct, short range patch-ruffle interactions are observed, but more common are extensive long range correlations that extend over the entire cell surface.