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.
Booth
M J
MJ
University of Oxford, Department of Engineering Science, Parks Road, Oxford OX1 3PJ, United Kingdom. martin.booth@eng.ox.ac.uk
Wilson
T
T
D008856:Microscopy, Fluorescence / Q000379:methods*; D008962:Models, Theoretical; D017785:Photons; D012031:Refractometry