Effects of specimen morphology on adaptive confocal and multiphoton microscopy

Abstract

Microscopy of thick biological specimens is often detrimentally affected by specimen-induced aberrations. In the simplest case, these aberrations arise from a refractive index mismatch between the immersion and mounting media. In other situations, the aberrations arise from variations in refractive index within the specimen. These aberrations cause loss of signal and reduced resolution. Aberrations can be corrected using adaptive optics, where a deformable mirror introduces equal but opposite aberrations into the optical path. Aberration correction can be performed by reconfiguring the deformable mirror and using the fluorescence signal as feedback, effectively maximising the fluorescence intensity. However, the degree to which aberrations affect the intensity is related to the distribution of fluorescence in the specimen. Signals from point-like objects are affected more than equivalent signals from from planar or volume objects. We investigate this effect and discuss the implications for adaptive optical microscopy of biological specimens.