The effect of refractive index mismatch on the image quality in two-photon confocal fluorescence microscopy is investigated by experiment and numerical calculations. The results show a strong decrease in the image brightness using high-aperture objectives when the image plane is moved deeper into the sample. When exciting at 740 nm and recording the fluorescence around 460 nm in a glycerol-mounted sample using a lens of a numerical aperture of 1.4 (oil immersion), a 25% decrease in the intensity is observed at a depth of 9 pm. In an aqueous sample, the same decrease is observed at a depth of 3 pm. By reducing the numerical aperture to 1.0, the intensity decrease can be avoided at the expense of the overall resolution and signal intensity. The experiments are compared with the predictions of a theory that takes into account the vectorial character of light and the refraction of the wavefronts according to Fermat's principle. Advice is given concerning how the effects can be taken into account in practice.

The effect of the specimen refractive index on the resolution, image brightness and axial scaling in confocal fluorescence microscopy is theoretically investigated for ten of the most popular fluorophores. The calculations are based on a vectorial theory based on Fermat's principle. We show that the axial scaling of the image does not depend on the fluorophore used. Axial scaling factors are derived for objects embedded in glycerol and water. For each fluorophore, the maximum intensity and the full‐width‐half‐maxima of the confocal point spread function are given as a function of the focusing depth. The full‐width‐half‐maxima allow for an assessment of the axial and lateral resolution when deeper regions of the specimen are to be investigated.

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A polarization interferometric method is presented for the quantitative microscopy of topographical structures with subwavelength linewidths. A liquid-crystal phase shifter is inserted into the imaging optics of a reflected-light microscope, and the principles of phase-shifting interferometry are applied to measuring the phase and the contrast of the TE-polarized image (E parallel edge) with the TM-polarized image (E perpendicular edge) as the reference. This common-path interferometric method provides selective edge detection for line structures because the polarization difference is localized at the structure edges. Two different threshold criteria for linewidth determination are discussed: distance of the contrast minima and distance of the points of the steepest phase change. Linewidths as small as 300 nm were measured at a 635-nm wavelength. The dependence on the illumination numerical aperture, as well as on the material, the width, and the depth of the structure, is investigated both experimentally and by rigorous numerical simulations.