Abstract
Background and Aims: Animal tissues are generally opaque. The opacity is derived from the higher refractive indexes of the tissue constituents, mainly the proteins and lipids, than that of the water molecules, causing scattering and creating difficulties in high-resolution deep-tissue microscopy. To reduce tissue scattering, in this research we aim to synthesize a high-refractive-index hydrogel for tissue permeabilization and embedding to prepare transparent gastrointestinal specimens for 3-dimensional (3-D) microscopy. Methods and Results: An aqueous solution of the monomers acrylamide and methacrylamide with the crosslinker triethylene glycol dimethacrylate is used to synthesize the AMEGA hydrogel with high refractive index (RI at 1.50) through ultraviolet polymerization. The optical-clearing capacity of AMEGA hydrogel is demonstrated by its compatibility with the lipids and proteins in solution (milk) and with tissue (intestinal and pancreatic biopsies), forming transparent gel-milk and gel-tissue complexes after ultraviolet radiation. Importantly, when the intestinal and pancreatic biopsies were labeled with fluorescent probes and examined by confocal microscopy, efficient fluorescence excitation and emission from the gel-tissue complex were achieved, facilitating in-depth 3-D visualization of the tissue microstructure with high definition. Conclusion: The combination of hydrogel-based optical clearing and 3-D microscopy for penetrative tissue imaging does not require microtome sectioning and provides a useful tool for 3-D presentation and analysis of the gastrointestinal tissues in an integrated fashion.