REFRACTIVE-INDEX-INDUCED ABERRATIONS IN 2-PHOTON CONFOCAL FLUORESCENCE MICROSCOPY

The effect of refractive index mismatch on the image quality in two-ph oton confocal fluorescence microscopy is investigated by experiment an d numerical calculations. The results show a strong decrease in the im age brightness using high-aperture objectives when the image plane is moved deeper into the sample. When exciting at 740 nm and recording th e 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 mu m. In an aqueous sample, the same decrease is observed at a depth of 3 mu m. By reducing the numer ical aperture to 1.0, the intensity decrease can be avoided at the exp ense of the overall resolution and signal intensity. The experiments a re compared with the predictions of a theory that takes into account t he vectorial character of light and the refraction of the wavefronts a ccording to Fermat's principle. Advice is given concerning how the eff ects can be taken into account in practice.