Development of a laser scanning microscope for simultaneous three-dimensional imaging in both a full-field laser scanning mode (FLSM) and a confocal laser scanning mode (CLSM) permits the direct comparison of axial resolution and out-of-focus background rejection as a function of sample thickness for both FLSM and CLSM with varying detector aperture (pinhole) radii. The sample-dependent detector aperture radii that optimize the signal-to-noise ratio (S/N) in the CLSM are experimentally determined. The results verify earlier calculations [ Appl. Opt. 33, 603 ( 1994)]. Using these results, we discuss the practical and theoretical limits on the S/N in the CLSM and compare them with those of a full-field epifluorescence microscope (FEM) that is enhanced by image deconvolution. The specimen volume over which the FLSM exhibits imaging properties that are equivalent to a FEM is calculated in the appendices.
© 1995 Optical Society of America
Original Manuscript: September 28, 1993
Revised Manuscript: July 27, 1994
Published: July 1, 1995
D. R. Sandison, D. W. Piston, R. M. Williams, and W. W. Webb, "Quantitative comparison of background rejection, signal-to-noise ratio, and resolution in confocal and full-field laser scanning microscopes," Appl. Opt. 34, 3576-3588 (1995)