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

J. Biomed. Opt., Vol. 6, Pt. 3, July 2001, 266-272. M. J. Booth & T. Wilson

 

We examine the effects of aberrations induced by a refractiveindex mismatch on the signal level and resolution of singlephoton(12p) and two-photon (22p), conventional and confocalscanning microscopes. In particular, we consider the aberrations introducedby an interface between oil/glass and water. Resolution isdefined in terms of enclosed fluorescence, rather than full-width halfmaximum,revealing more useful information for heavily aberratedpoint spread functions (PSFs). It is shown that, at large focusingdepths, the resolution of 22p conventional and 12p confocal microscopesare almost identical. The benefits of aberration correctionare examined by removing Zernike aberration modes. With aberrationcorrection, the best resolution is found for 12p confocal and 22pconfocal modes. An approximation based upon geometrical optics isalso introduced which shows that the axial resolution of heavily aberratedPSFs is roughly proportional to focusing depth.


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