ABERRATIONS IN CONFOCAL FLUORESCENCE MICROSCOPY INDUCED BY MISMATCHESIN REFRACTIVE-INDEX

The effect of refractive-index mismatch, as encountered in the observa tion of biological specimens, on the image acquisition process in conf ocal fluorescence microscopy is investigated theoretically. The analys is takes the vectorial properties of light into account and is valid f or high numerical apertures. Quantitative predictions on the decrease of resolution, intensity drop and shift of focus are given for practic al situations. When observing with a numerical aperture of 1.3 (oil im mersion) and an excitation wavelength of 514 nm the centre of the focu s shifts 1.7 mum per 10 mum of axial displacement in an aqueous medium , thus yielding an image that is scaled by a factor of 1.2 in the axia l direction. Furthermore, it can be expected that for a fluorescent pl ane 20 mum deep inside an aqueous medium the peak intensity is 40% les s than for a plane which is 10 mum deep. In addition, the axial resolu tion is decreased by a factor of 1.4. The theory was experimentally ve rified for test samples with different refractive indices.