Abstract
Inhomogeneities in the refractive index of a biological microscopy sample can introduce phase aberrations, 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 highly heterogeneous nature of biological tissues, the 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, in a significantly shorter time compared to sequential isoplanatic correction of multiple image subregions. The method was tested in whole Drosophila brains and in larval Zebrafish, each showing a dramatic improvement in resolution and sharpness when compared to conventional isoplanatic adaptive optics.
Highlights
Adaptive optics(AO) is a method of growing popularity for the correction of phase aberrations in optical systems
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, in a significantly shorter time compared to sequential isoplanatic correction of multiple image subregions
This paper presents a novel approach to anisoplanatic correction in laser scanning confocal fluorescence microscopy, allowing for spatially varying aberration correction of the excitation light throughout the field of view by parallelized acquisition of multiple isoplanatic patches with an array of independently corrected foci generated by a spatial light modulator (SLM)
Summary
Adaptive optics(AO) is a method of growing popularity for the correction of phase aberrations in optical systems. Optical microscopes can benefit from AO, as most samples of interest for life sciences are thick and inhomogeneous, introducing significant phase aberrations to light propagating through them. In the classical implementation of AO [2], an adaptive optical element (AOE), such as a deformable mirror, or a liquid crystal spatial light modulator, is generally introduced in the back aperture plane of the system. The main limitation in this approach is due to the fact that a AOE in the pupil plane of an optical system is only capable of applying an isoplanatic correction, meaning the same two-dimensional pupil plane correction is applied to all points within the field of view. The anisoplanatic nature of sample-induced aberrations requires independent correction for each point of the field of view, which can not be achieved with a single AOE, as schematically represented, panel a The anisoplanatic nature of sample-induced aberrations requires independent correction for each point of the field of view, which can not be achieved with a single AOE, as schematically represented in Fig. 1, panel a
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