Anisoplanatic adaptive optics in parallelized laser scanningmicroscopy
Abstract
Inhomogeneities in the refractive index of a biological sample can introduce phase aberrationsin microscopy systems, severely impairing the quality of images. Adaptive optics can be employed to correct for phase aberrations and improve image quality. However, conventional adaptive optics can only correct a single phase aberration for the whole field of view (isoplanatic correction) while, due to the three dimensional nature of biological tissues, sample induced aberrations in microscopy often vary throughout the field of view (anisoplanatic aberration), limiting significantly the effectiveness of adaptive optics. This paper reports on a new approach for aberration correction in laser scanning confocal microscopy, in which a spatial light modulator is used to generate multiple excitation points in the sample to simultaneously scan different portions of the field of view with completely independent correction, achieving anisoplanatic compensation of sample induced aberrations. The method was tested in 150 μm thick whole Drosophila brains, showing a dramatic improvement in resolution and sharpness when compared to conventional isoplanatic adaptive optics.