On-the-fly deconvolution of diagonally-scanned Airy lightsheet microscopy

Abstract

Its subcellular resolution and minimal sample exposure make light-sheet microscopy the ideal tool to study biological specimen during their early development. A light-sheet microscope scans the sample with a plane of light and collects fluorescence with an objective orthogonal to the illumination. However, tightly focused Gaussian light-sheets suffer from a shallow depth of focus and are susceptible to scattering-induced aberrations. Light-sheets created by non-diffractive Airy beams can overcome this to yield isotropic sub-cellular resolution over a ten-fold larger field-of-view. Airy beam light-sheets have a characteristically curved structure and a broad transverse structure with side-lobes. Digital deconvolution of the raw data is thus essential to obtain highfidelity images. Provided that the scan is along the direction of the detection axis, and all recorded data fits within working memory, a simple and efficient Wiener filter can recover accurate 3D images. However, multimillimeter sized samples must be scanned with a light-sheet that is diagonal to the sample surface. The diagonal movement prevents the use of standard Wiener filtering. Moreover, the associated data sets can become too large to fit within the working memory of a consumer-grade GPU. This demands slow off-line processing, thus breaking a rapid experimental feedback-loop. Here, we investigate the potential of on-the-fly deconvolution of diagonally-scanned Airy light-sheet microscopy.