Highly Sensitive Shack-Hartmann Wavefront Sensor: Application to Non-Transparent Tissue Mimic Imaging with Adaptive Light-Sheet Fluorescence Microscopy.

Brajones Brajones,Dovillaire Dovillaire,Clouvel Clouvel,Lorenzo Lorenzo,Levecq Levecq

doi:10.3390/mps2030059

Abstract

High-quality in-depth imaging of three-dimensional samples remains a major challenge in modern microscopy. Selective plane illumination microscopy (SPIM) is a widely used technique that enables imaging of living tissues with subcellular resolution. However, scattering, absorption, and optical aberrations limit the depth at which useful imaging can be done. Adaptive optics (AOs) is a method capable of measuring and correcting aberrations in different kinds of fluorescence microscopes, thereby improving the performance of the optical system. We have incorporated a wavefront sensor adaptive optics scheme to SPIM (WAOSPIM) to correct aberrations induced by optically-thick samples, such as multi-cellular tumor spheroids (MCTS). Two-photon fluorescence provides us with a tool to produce a weak non-linear guide star (NGS) in any region of the field of view. The faintness of NGS; however, led us to develop a high-sensitivity Shack–Hartmann wavefront sensor (SHWS). This paper describes this newly developed SHWS and shows the correction capabilities of WAOSPIM using NGS in thick, inhomogeneous samples like MCTS. We report improvements of up to 79% for spatial frequencies corresponding to cellular and subcellular size features.

Highlights

In recent years, tissue mimics (TMs) such as microtissues, spheroids, and organoid cultures have become increasingly important in life-science research, as they provide a physiologically more relevant environment for cell growth, tissue morphogenesis, and stem cell differentiation
By coupling our wavefront sensor adaptive optics scheme to SPIM (WAOSPIM) with an Electron multiplying CCD (EMCCD)-based SHWFS designed for TM imaging, we demonstrate the in-depth correction capabilities in multi-cellular tumor spheroids (MCTS) up to 120 μm deep
We opted for a non-linear guide star approach, using fluorescent labeled proteins to create a point-like source of light through two-photon excitation

Summary

Introduction

Tissue mimics (TMs) such as microtissues, spheroids, and organoid cultures have become increasingly important in life-science research, as they provide a physiologically more relevant environment for cell growth, tissue morphogenesis, and stem cell differentiation. Bead placement inside living samples is random and intrusive [13], and requires the use of a pinhole limiting the usefulness of the correction To overcome these difficulties and to provide a minimally-invasive and more versatile approach, Aviles-Espinosa [8] and Wang [14] proposed the use of a non-linear guide star (NGS) that was successfully applied to confocal [15] and lattice light-sheet microscopes [7]. In samples such as MCTS, the NGS fluorescence light reaching the sensor is weak and often limits the accuracy of the wavefront measurement and; the efficiency of the correction. To be able to work with minimal light, the SHWFS development focused on photon collection efficiency, optimizing the microlens array to maximize

Methods

Experimental Setup

Image Processing

Findings

Discussion and Conclusions