Use of High-Refractive Index Hydrogels and Tissue Clearing for Large Biological Sample Imaging

Recent advances in tissue clearing and light sheet fluorescence microscopy have improved insights into and understanding of tissue morphology and disease pathology by imaging large samples without the requirement of histological sectioning. However, sample handling and conservation of sample integrity during lengthy staining and acquisition protocols remains a challenge. This study overcomes these challenges with acrylamide hydrogels synthesised to match the refractive index of solutions typically utilised in aqueous tissue clearing protocols. These hydrogels have a high-water content (82.0 ± 3.7% by weight). The gels are stable over time and FITC-IgG readily permeated into and effluxed out of them. Whilst the gels deformed and/or swelled over time in some commonly used solutions, this was overcome by using a previously described custom refractive index matched solution. To validate their use, CUBIC cleared mouse tissues and whole embryos were embedded in hydrogels, stained using fluorescent small molecule dyes, labels and antibodies and successfully imaged using light sheet fluorescence microscopy. In conclusion, the high water content, high refractive index hydrogels described in this study have broad applicability to research that delves into pathophysiological processes by stabilising and protecting large and fragile samples.

Light Sheet Fluorescence Microscopy Illuminating Soft Matter

Volumetric microscopic imaging data acquired at high speeds is often needed in studies of soft matter. Several microscopy techniques exist for this purpose, but a relative newcomer is light sheet fluorescence microscopy (LSFM). This microscopy method has seen spectacular growth in the biological sciences over the past two decades. In this perspective, we highlight how LSFM may also apply to the field of soft matter. We review the principles and recent advances of LSFM and discuss how it has been used in prior soft matter studies. We demonstrate how a recent implementation of LSFM can be used to study capillary wave fluctuations and droplet coalescence in a colloidal fluid system.