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Aberrations are known to severely compromise image quality in optical microscopy, especially when high numerical aperture (NA) lenses are used in confocal fluorescence microscopy (CFM) and two-photon microscopy (TPM). The method of adaptive optics may correct aberrations and restore diffraction limited operation. So far the problem of aberrations that occur in the imaging of biological specimens has not been quantified. However, this information is essential for the design of adaptive optics systems. We have therefore built an interferometer incorporating high NA objective lenses to measure the aberrations introduced by biological specimens. The measured wavefronts were decomposed into their Zernike mode content in order both to classify and quantify the aberrations. We calculated the potential benefit of correcting different numbers of Zernike modes using different NAs in an adaptive CFM by comparing the signal levels before and after correction. The results indicate that adaptive correction of low order Zernike modes can provide significant benefit for many specimens. The results also show that quantitative fluorescence microscopy may be strongly affected by specimen induced aberrations in non-adaptive systems.

Characterizing specimen induced aberrations for high NA adaptive optical microscopy