Dual-view light-sheet imaging through tilted glass interface using a deformable mirror

Abstract

Light-sheet microscopy has become one of the primary tools for imaging live developing organisms because of its high speed, low phototoxicity, and optical sectioning capabilities. Detection from multiple sides (multi-view imaging) additionally allows nearly isotropic resolution via computational merging of the views. However, conventional light-sheet microscopes require that the sample is suspended in a gel to allow optical access from two or more sides. At the same time, the use of microfluidic devices is highly desirable for many experiments, but geometric constrains and strong optical aberrations caused by the coverslip titled relative to objectives make the use of multi-view lightsheet challenging for microfluidics. In this paper we describe the use of adaptive optics (AO) to enable multi-view light-sheet microscopy in such microfluidic setup by correcting optical aberrations introduced by the tilted coverslip. The optimal shape of deformable mirror is computed by an iterative stochastic gradient-descent algorithm that optimizes PSF in two orthogonal planes simultaneously. Simultaneous AO correction in two optical arms is achieved via a knife-edge mirror that splits excitation path and combines the detection path. We characterize the performance of this novel microscope setup and, by dual-view light-sheet imaging of C.elegans inside a microfluidic channel, demonstrate a drastic improvement of image quality due to AO and dual-view reconstruction. Our microscope design allows multi-view light-sheet microscopy with microfluidic devices for precisely controlled experimental conditions and high-content screening.