A Novel Fast Volumetric Light Sheet Microscopy

Abstract

Fast noninvasive three-dimensional (3D) imaging is crucial for the quantitative understanding of highly dynamic biological processes. Over the last decades, several fluorescence microscopy techniques have been developed in order to provide a faster and deeper imaging of thick biological samples [1]. Within this framework, Light Sheet Fluorescence Microscopy (LSFM) has emerged as a powerful imaging tool for 3D imaging of thick samples ranging from single cells to entire animals [2,3].However, to obtain a 3D reconstruction either sample or microscope parts usually need to be moved limiting the acquisition speed and inducing possible interferences in volume recording. To solve this problem, herein we propose a new light-sheet-based optical scheme that enables fast volumetric recording at unprecedented temporal resolution. 3D imaging speeds up to 100 Volumes per Second (faster than three times the volumetric video rate) have been achieved without sample rotation or translation. Volumetric acquisition speed is limited only by the acquisition frame rate of the CCD camera and by a reasonable signal to noise ratio. Our optical approach allows invariant acquisition along the detection axis avoiding extensive processing and deconvolution methods to restore image quality. We demonstrate imaging performance of the microscope by fast volumetric acquisition of flowing beads and live Paramecium movements.1. Diaspro A. Nanoscopy and Multidimensional Optical Fluorescence Microscopy, Chapman and Hall/CRC, (2010).2. E.H. Stelzer. Light-sheet fluorescence microscopy for quantitative biology. Nat Methods. 12(1):23-6. (2014).3. Huisken J and Stainier DYR. Selective plane illumination microscopy techniques in developmental biology. Development, 136(12):1963-1975, (2009).