THEORETICAL-ANALYSIS OF A ROTATING-DISK PARTIALLY CONFOCAL SCANNING MICROSCOPE

Confocal scanning microscopy is widely used for three-dimensional (3-D ) visualization of fixed specimens but has found only a limited 3-D re construction application for living specimens because the high intensi ty of the excitation often damages the specimen or causes the fluoresc ent dye to bleach. Computational optical-sectioning microscopy also su ffers from drawbacks because nonconfocal 3-D imaging is fundamentally constrained by an artifactual elongation in the optical axis imposed b y the so-called missing cone. We investigate the imaging properties of a new rotating-disk partially confocal scanning microscope (PCSM) tha t greatly reduces collection time by using multiple apertures for both excitation and detection, effectively working as many confocal micros copes in parallel. We show that this PCSM behaves as a hybrid microsco pe; near the in-focus plane it behaves near the theoretical optimum fo r confocal microscopy, and away from this plane its behavior approache s that of a nonconfocal microscope. We also show that the rotating-dis k PCSM does not suffer from a missing cone. In fact, the optical trans fer function of the theoretically optimal confocal microscope and the rotating-disk PCSM have practically the same bandpass in the spatial-f requency domain.