Specimen-induced aberrations and adaptive optics for microscopy

Proc. SPIE Vol. 5894, Advanced Wavefront Control: Methods, Devices, and Applications III, August 2004, 589403.

The imaging properties of optical microscopes can be severely compromised by specimen-induced aberrations causing degraded resolution, reduced signal levels, and image distortion. This is particularly the case in highresolution,three-dimensional techniques, such as scanning confocal or multi-photon  fluorescence microscopy -techniques used extensively in the biological sciences. The aberrations are caused by spatial variations of refractiveindex within the specimen itself. In wide-eld microscopes, this gives rise to aberrations that change across the field of view; in scanning microscopes, they cause temporal variations as the focal spot is scanned through the specimen. The application of adaptive optics to this problem has obvious potential and the principle has been demonstrated in scanning microscopes. To characterise the optical properties of specimens and determine the requirements for adaptive microscopes, we have performed the rst detailed study of biological specimen-induced aberrations using an interferometer incorporating high NA microscope objectives. We show that low order correction of aberrations produces signicant recovery of signal and resolution and we compare the performance of dierent correction devices, e.g. deformable and segmented mirrors, for imaging such specimens. It is also shown that that the presence of tip, tilt and defocus modes leads to three-dimensional image distortion that is

not easily removed by an adaptive correction system.


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