Size and Shape of The Confocal Spot: Control and Relation to 3D Imaging and Image Processing

Abstract

A confocal microscope can be considered as a 3D sampling instrument for collecting data from spatial structures, especially biological ones. Optimal data collection in confocal microscopes requires the adaptation of the dimensions of the sampling volume to the lateral and axial raster parameters employed during data collection. It is shown how, in principle, the collection volume can be partly manipulated by the use of variable pinholes both in the illumination and detection paths. The effective confocal spot will depend on the optics used, the degree of aberration, and the alignment of the instrument. Measurements of the axial response both in fluorescence and reflection for some high N.A. lens systems as a function of the above factors are presented. The use of variable pinholes in computer-controlled instruments is discussed, especially in relation to operation in fluorescence. It is indicated that proper interpretation and processing of 3D confocal data requires at least approximate knowledge of the applicable 3D response function.