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 mismatc h on the signal level and resolution of single-photon (1 - p) and two-photo n (2 - p), conventional and confocal scanning microscopes. In particular, w e consider the aberrations introduced by an interface between oil/glass and water. Resolution is defined in terms of enclosed fluorescence, rather tha n full-width half-maximum, revealing more useful information for heavily ab errated 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 b y removing Zernike aberration modes. With aberration correction, the best r esolution is found for 1 - p confocal and 2-p confocal modes. An approximat ion based upon geometrical optics is also introduced which shows that the a xial resolution of heavily aberrated PSFs is roughly proportional to focusi ng depth. (C) 2001 Society of Photo-Optical Instrumentation Engineers.