Experimental comparisons of confocal and nonconfocal scanning microscopes

We have performed direct experimental comparisons between confocal and nonconfocal microscopes, using an apparatus that functions simultaneously in both modes. A subresolution pinhole illuminated by a focused laser beam is imaged by a microscope objective onto an object, which is mechanically scanned. A portion of the scattered light is captured by the objective, on the long side of which are two pellicle beam splitters. After one is a confocal detection arrangement, consisting of a pinhole followed by a detector. The other beam splitter is followed by a nonconfocal arrangement, an adjustable finite aperture and a detector. The two images are formed simultaneously, with every aspect of the system identical, except for the detector configurations.