Abstract
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.
Highlights
The use of histological approaches to understand physiology and pathology has traditionally relied on optical imaging modalities
Synthesis of High Refractive Index Hydrogels tri(ethylene glycol) dimethylacrylate (TEDA) (Figure 1C) [19] were synthesised and Hydrogels composed of acrylamide (Figure 1A), methacrylamide (Figure 1B) and uatedtri(ethylene for size changes (Figure 1D) and water content (Figure 1E)
We evaluated the applicability of a high refractive index hydrogel that provided stability to tissue architecture and minimised shrinking/expanding of the tissue for use in imaging large biological samples with light sheet fluorescence microscopy (LSFM) [6,21]
Summary
The use of histological approaches to understand physiology and pathology has traditionally relied on optical imaging modalities. The opaque nature of many biological samples is a major limitation to this approach. Serial sectioning can be used to overcome this, with light scattering significantly decreased when tissues are sectioned into slices less than 100 μm thick. A major drawback to this approach is that the original 3-dimensional tissue architecture is not preserved. Whilst algorithms that automatically align serial sections can be utilised, sectioning tissue always leads to non-linear distortion. The advent of tissue clearing and whole-mount imaging systems such as light sheet fluorescence microscopy (LSFM) effectively overcomes this limitation, facilitating the visualisation of cell-to-cell interactions within complex 3-dimensional tissue architectures
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