Refractive-index-mismatch induced aberrations in single-photon and two-photon microscopy and the use of aberration correction

We examine the effects of aberrations induced by a refractive index mismatch on the signal level and resolution of single-photon (1 - p) and two-photon (2 - p), conventional and confocal scanning microscopes. In particular, we consider the aberrations introduced by an interface between oil/glass and water. Resolution is defined in terms of enclosed fluorescence, rather than full-width half-maximum, revealing more useful information for heavily aberrated point spread functions (PSFs). It is shown that, at large focusing depths, the resolution of 2 - p conventional and 1 - p confocal microscopes are almost identical. The benefits of aberration correction are examined by removing Zernike aberration modes. With aberration correction, the best resolution is found for 1 - p confocal and 2-p confocal modes. An approximation based upon geometrical optics is also introduced which shows that the axial resolution of heavily aberrated PSFs is roughly proportional to focusing depth. (C) 2001 Society of Photo-Optical Instrumentation Engineers.