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We present an overview of recent theories for describing specimen-induced spherical aberration in confocal microscopy. One of these theories is used to compute numerically the role of spherical aberration in general confocal, and especially in biological confocal, microscopy for a variety of three-layer specimen structures. In particular, we study the effect of specimen-induced spherical aberration on the maximum value of the overall confocal point spread function, the accompanying focal shift and the size of the optical probe in both fluorescence and brightfield confocal microscopy.

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K E Y w ORDS . Scanning microscopy, confocal systems, image formation, confocal design. SUMMARYWe consider the imaging in scanning microscopes employing point and slit-shaped detectors in both the bright-field and fluorescent mode. In particular we consider the three-dimensional aspects of the imaging and show inter alza that acceptable, albeit asymmetrical, images result from a slit detector system at low levels of defocus. The situation becomes worse as the defocus is increased although acceptable extendedfocus and isometric images are still possible in some cases.