Adaptive aberration correction in a confocal microscope

Proc. Nat. Acad. Sci., Vol. 99, No. 9, 30 April 2002, 5788-5792. M. J. Booth, M. A. A. Neil, R. Juskaitis & T. Wilson

The main advantage of confocal microscopes over their conventionalcounterparts is their ability to optically ‘‘section’’ thickspecimens; the thin image slices thus obtained can be used toreconstruct three-dimensional images, a capability which is particularlyuseful in biological applications. However, it is well knownthat the resolution and optical sectioning ability can be severelydegraded by system or specimen-induced aberrations. The use ofhigh aperture lenses further exacerbates the problem. Moreover,aberrations can considerably reduce the number of photons thatreach the detector, leading to lower contrast. It is rather unfortunate,therefore, that in practical microscopy, aberration-free confocalimaging is rarely achieved. Adaptive optics systems, whichhave been used widely to correct aberrations in astronomy, offera solution here but also present new challenges. The optical systemand the source of aberrations in a confocal microscope are considerablydifferent and require a novel approach to wavefrontsensing. This method, based upon direct measurement of Zernikeaberration modes, also exhibits an axial selectivity similar to thatof a confocal microscope. We demonstrate an adaptive confocalfluorescence microscope incorporating this modal sensor togetherwith a deformable membrane mirror for aberration correction.Aberration corrected images of biological specimens show considerableimprovement in contrast and apparent restoration of axialresolution.
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