Quantitative comparison of background rejection, signal-to-noise ratio, and resolution in confocal and full-field laser scanning microscopes

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.