Characterizing specimen induced aberrations for high NA adaptive optical microscopy

Opt. Express Vol. 12, December 2004, pp. 6540-6552. M. Schwertner, M. J. Booth, and T. Wilson
Aberrations are known to severely compromise image qualityin optical microscopy, especially when high numerical aperture (NA) lensesare used in confocal fluorescence microscopy (CFM) and two-photonmicroscopy (TPM). The method of adaptive optics may correct aberrationsand restore diffraction limited operation. So far the problem of aberrationsthat occur in the imaging of biological specimens has not been quantified.However, this information is essential for the design of adaptive opticssystems. We have therefore built an interferometer incorporating high NAobjective lenses to measure the aberrations introduced by biological specimens.The measured wavefronts were decomposed into their Zernike modecontent in order both to classify and quantify the aberrations. We calculatedthe potential benefit of correcting different numbers of Zernike modes usingdifferent NAs in an adaptive CFM by comparing the signal levels beforeand after correction. The results indicate that adaptive correction of loworder Zernike modes can provide significant benefit for many specimens.The results also show that quantitative fluorescence microscopy may bestrongly affected by specimen induced aberrations in non-adaptive systems


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