IMAGE SHARPNESS AND CONTRAST TRANSFER IN COHERENT CONFOCAL MICROSCOPY

Confocal microscopes provide clear, thin optical sections with little disturbance from regions of the specimen that are not in focus. In add ition, they appear to provide somewhat greater lateral and axial image resolution than with nonconfocal microscope optics. To address the qu estion of resolution and contrast transfer of light microscopes, a new test slide that enables the direct measurement of the contrast transf er characteristics (CTC) of microscope optics at the highest numerical aperature has been developed. With this new test slide, the performan ce of a confocal scanning laser microscope operating in the confocal r eflection mode and the non-confocal transmission mode was examined. Th e CTC curves show that the confocal instrument maintains exceptionally high contrast (up to twice that with non-confocal optics) as the dime nsion of the object approaches the diffraction limit of resolution; at these dimensions, image detail is lost with non-confocal microscopes owing to a progressive loss of image contrast. Furthermore, we have ca lculated theoretical CTC curves by modelling the confocal and non-conf ocal imaging modes using discrete Fourier analysis. The close agreemen t between the theoretical and experimental CTC curves supports the ear lier prediction that the coherent confocal and the incoherent non-conf ocal imaging mode have the same limit of resolution (defined here as t he inverse of the spatial frequency at which the contrast transfer con verges to zero). The apparently greater image resolution of the cohere nt confocal optics is a consequence of the improved contrast transfer at spacings which are close to the resolution limit.