Measurement of specimen induced aberrations of biological samples using phase stepping interferometry

J. Microsc., Vol. 213, Pt. 1, January 2004, 11-19. M. Schwertner, M.J. Booth, M.A.A. Neil & T. Wilson.

Confocal or multiphoton microscopes, which deliver opticalsections and three-dimensional (3D) images of thick specimens,are widely used in biology. These techniques, however, aresensitive to aberrations that may originate from the refractiveindex structure of the specimen itself. The aberrations causereduced signal intensity and the 3D resolution of the instrument is compromised. It has been suggested to correct foraberrations in confocal microscopes using adaptive optics. Inorder to define the design specifications for such adaptive opticssystems, one has to know the amount of aberrations presentfor typical applications such as with biological samples. Wehave built a phase stepping interferometer microscope thatdirectly measures the aberration of the wavefront. The modalcontent of the wavefront is extracted by employing Zernikemode decomposition. Results for typical biological specimensare presented. It was found for all samples investigated thathigher order Zernike modes give only a small contribution tothe overall aberration. Therefore, these higher order modes canbe neglected in future adaptive optics sensing and correctionschemes implemented into confocal or multiphoton microscopes, leading to more efficient designs.
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