Three-dimensional (3D) imaging deep within biological tissues is a significant challenge in biology due to light scattering caused by the optical heterogeneity of tissue components. Traditional sectioning methods are laborious and can distort tissue. To overcome this, various 3D tissue clearing techniques have emerged, broadly categorized as organic solvent-based and aqueous-based. Organic solvent-based methods, while achieving rapid transparency, often cause significant tissue shrinkage and fluorescence loss. Aqueous-based methods generally preserve fluorescence and tissue size better but can suffer from slow clearing rates, insufficient transparency, or complex procedures. Examples include methods based on high refractive index (RI) solutions like 2,2'-thiodiethanol, hyperhydration with urea and lipid removal, or hydrogel embedding with lipid removal (e.g., CLARITY). Each approach has limitations, such as long incubation times, poor tissue penetration, or incompatibility with lipophilic dyes. This research addresses the need for a single, optimized solution that overcomes the shortcomings of existing methods.

The existing literature extensively documents various optical clearing techniques for 3D tissue imaging. Organic solvent-based methods like BABB (Benzoic Acid Benzyl Benzoate), dibenzyl ether, 3DISCO (3D imaging of solvent-cleared organs), and UDISCO (ultimate DISCO) offer fast clearing but significant tissue shrinkage and fluorescence quenching. Aqueous-based methods, including those using 2,2'-thiodiethanol, SeeDB (SeeDeepBrain), FocusClear, RIMS (Refractive Index Matching Solution), Scale, ScaleS, CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis), and FRUIT (combining SeeDB and urea) have been developed to address these issues. However, challenges such as long clearing times, insufficient transparency for thick tissues, loss of protein content, and procedure complexity persist. CLARITY and its variants utilize hydrogel embedding and electrophoretic lipid removal, achieving high transparency but requiring specialized equipment and incompatibility with lipophilic dyes. MACS (m-xylylenediamine-based Aqueous Clearing System) shows improved compatibility with lipophilic dyes but has moderate transparency. This research aimed to develop an optimized method that surpasses these limitations.

The researchers developed OptiMuS by combining optimized concentrations of urea, iohexol (a high RI, low viscosity substance), and D-sorbitol. Urea promotes hyperhydration and tissue penetration, while D-sorbitol aids in gentle clearing and size preservation. The optimal concentrations were determined experimentally, balancing transparency with minimal tissue deformation. The researchers tested OptiMuS on various tissues (rat and mouse brain, liver, lung, intestine, heart, kidney, and spleen) and compared its performance against existing methods including CLARITY, CUBIC, ScaleS, ScaleSQ(0), MACS, and FOCM (FocusClear). Comparisons included clearing time, transparency (measured using spectrophotometry and bright-field imaging), and size preservation (quantified using ImageJ). Fluorescence preservation was assessed using Thy1-EYFP transgenic mice brains, measuring fluorescence intensity over time. Image quality was evaluated using confocal and light-sheet fluorescence microscopy (LSFM), including signal-to-noise ratio (SNR) measurements. OptiMuS's compatibility with lipophilic dyes (DiI) was demonstrated by labeling vascular structures in whole brains, kidneys, spleens, and intestines. Finally, 3D quantitative analysis of glomerular structures in normal and diseased (nephrotoxic nephritis, NTN) kidneys was performed using OptiMuS combined with DXplorer, a 3D morphological analysis software, and validated by manual measurements using MeshLab.

OptiMuS demonstrated superior performance compared to other aqueous-based clearing methods. It achieved high transparency (~75%) in 1-mm thick rat brain tissue within 1.5 hours with negligible size change (0.93 ± 1.1% shrinkage). It significantly outperformed other methods in terms of transparency across a wide wavelength range (400-800 nm) and maintained superior transparency with minimal size change compared to other methods. OptiMuS effectively preserved endogenous EYFP fluorescence signals (over 90% after 4 days), significantly better than CLARITY and FOCM. Confocal imaging showed excellent preservation of neuronal ultrastructure and high SNR values throughout the entire depth of 1-mm thick tissue. LSFM imaging successfully visualized fine neural structures in whole mouse brains from Thy1-EYFP transgenic mice and cholinergic neurons in ChAT-Cre-tdTomato transgenic mice brains and intestines. OptiMuS was also compatible with immunostaining and visualization of various cellular structures with high resolution. Crucially, OptiMuS preserved DiI signals in vascular structures of whole organs (brain, intestine, spleen), enabling detailed 3D visualization of vascular networks. Finally, using OptiMuS with DXplorer software, the researchers performed 3D quantitative analysis of glomerular structures in normal and NTN model mouse kidneys, revealing significant differences in glomerulus volume and morphology between the groups. The accuracy of DXplorer's automated measurements was validated using manual measurements with MeshLab.

OptiMuS addresses the limitations of existing optical clearing methods by offering a simple, rapid, single-step approach with high transparency and minimal tissue distortion. Its compatibility with various labeling techniques, including lipophilic dyes, broadens its applicability. The superior performance in preserving both tissue structure and fluorescence signals makes it suitable for various imaging modalities and quantitative analysis. The successful application to diverse organs and disease models highlights its versatility and potential in various research areas, including neurobiology, nephrology, and pathology. The integration with DXplorer opens up possibilities for automated, quantitative analysis of complex 3D structures.

OptiMuS represents a significant advancement in optical clearing techniques. Its speed, simplicity, and effectiveness in preserving tissue integrity and fluorescence signals make it a powerful tool for 3D volume imaging across various biological applications. Future research could focus on optimizing OptiMuS for specific organs beyond the brain and integrating it with other imaging modalities, such as optical coherence tomography (OCT), for label-free 3D histopathology.

While OptiMuS demonstrated excellent performance in brain tissue, the degree of deformation was slightly higher in other organs, suggesting the need for organ-specific optimization of the solution composition and incubation time. The study primarily focused on mouse and rat tissues; further investigation is needed to determine its effectiveness in other species and tissue types. The long-term effects of OptiMuS on tissue preservation and potential artifacts from the clearing process warrant further investigation.