Aberration Correction for Confocal Imaging in Refractive Index Mismatched Media

J. Microsc., Vol. 192, Pt. 2, Nov. 1998, 90-98. M.J. Booth, M.A.A. Neil & T. Wilson

A major limitation to the use of confocal microscopes toimage thick biological tissue lies in the dramatic reductionin both signal level and resolution when focusing deep intoa refractive-index-mismatched specimen. This limitationmay be overcome by measuring the wavefront aberrationand pre-shaping the input beam so as to cancel the effects ofaberration. We consider the images of planar and pointobjects in brightfield, single-photon fluorescence and twophotonfluorescence imaging. In all cases, the specimensare imaged using an oil-immersion objective throughvarious thicknesses of water.The question of finite-sized pinhole is addressed and it isfound, in general, that it is sufficient to correct only the firsttwo or three orders of spherical aberration to restoreadequate image signal level and optical resolution, atimaging depths of up to 50–100 wavelengths.

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