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The effect of refractive-index mismatch, as encountered in the observation of biological specimens, on the image acquisition process in confocal fluorescence microscopy is investigated theoretically. The analysis takes the vectorial properties of light into account and is valid for high numerical apertures. Quantitative predictions on the decrease of resolution, intensity drop and shift of focus are given for practical situations. When observing with a numerical aperture of 1.3 (oil immersion) and an excitation wavelength of 514 nm the centre of the focus shifts 1.7 mu m per 10 mu m of axial displacement in an aqueous medium, thus yielding an image that is scaled by a factor of 1.2 in the axial direction. Furthermore, it can be expected that for a fluorescent plane 20 µ m deep inside an aqueous medium the peak intensity of 40% less for a plane which is 10 µ m deep. In addition, the axial resolution is decreased by a factor of 1.4. The theory was experimentally verified for test samples with different refractive indices.
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