Outlook on optimizing ultramicroscopy imaging technique through optical characterization

Abstract

Here, we present an optically optimized system for static ultramicroscopy imaging technique. The unit for generating an ultra-thin light sheet employs aspheric and meso-optical elements (meso-aspheric system). An analytical as well as an experimental comparison between the light sheet produced by the standard system (using a rectangular slit aperture and one cylindrical lens) and the one produced by our latest optimized system, which converts a symmetrical Gaussian beam into an ultra-thin light sheet is presented. Using the new light sheet in combination with our objective equipped with a modulator unit to compensate the refractive index mismatch between air and mediums with indices of 1.45-1.56, we present high resolution images of various biological samples that were chemically cleared using different methods. They demonstrate a marked improvement in quality, contrast and resolution.

High‐resolution imaging of fluorescent whole mouse brains using stabilised organic media (sDISCO)

“…Imaging was performed using a modified version of the ultramicroscopy setup as described . This modified system is equipped with two Sapphire lasers (Coherent Inc., Germany) emitting a 488 and 532 nm continuous Gaussian beam for fluorescence excitation and a custom‐made light sheet generator ). The data for Figure S4 were measured using a standard ultramicroscopy setup as described .…”

“…Recording was done using an Andor Neo CMOS camera (Andor, Ireland) with 2560 x 2160 pixel resolution. Microscopy was done using Olympus objectives (XLFLUOR 2x NA = 0.14, XLFLUOR 4x NA = 0.28, and LUCPLFLN 20x NA = 0.45), corrected to the RI of ~1.56 by custom‐made modulator units or a 25x objective (XLSLPlan N, NA = 1.0, Olympus, Tokyo, Japan) with integrated RI adjustment. We used a 525/50 nm filter for EGFP, a 550/49 nm filter for YFP and a 605/70 nm filter for tdTomato.…”

Introduction to the special section on light sheet fluorescence illumination microscopy

“…Furthermore, the optimization of Ultramicroscopy, thanks to the use of aspherical optical elements that are able to manipulate the phase and related factors, represents a step forward in the resolution improvement when tissue imaging is performed (Saghafi et al, ). This approach shows superior 3D imaging capabilities in tissues when combined with optical clearing methods.…”