Optimized single-step optical clearing solution for 3D volume imaging of biological structures

Abstract

OptiMuS is an optical clearing method which preserves endogenous fluorescence and sample sizes and can be used to clear thick tissues, visualize neural networks and vascular structures and diagnose pathological status of kidneys, as a potential application. Various optical clearing approaches have been introduced to meet the growing demand for 3D volume imaging of biological structures. Each has its own strengths but still suffers from low transparency, long incubation time, processing complexity, tissue deformation, or fluorescence quenching, and a single solution that best satisfies all aspects has yet been developed. Here, we develop OptiMuS, an optimized single-step solution that overcomes the shortcomings of the existing aqueous-based clearing methods and that provides the best performance in terms of transparency, clearing rate, and size retention. OptiMuS achieves rapid and high transparency of brain tissues and other intact organs while preserving the size and fluorescent signal of the tissues. Moreover, OptiMuS is compatible with the use of lipophilic dyes, revealing DiI-labeled vascular structures of the whole brain, kidney, spleen, and intestine, and is also applied to 3D quantitative and comparative analysis of DiI-labeled vascular structures of glomeruli turfs in normal and diseased kidneys. Together, OptiMuS provides a single-step solution for simple, fast, and versatile optical clearing method to obtain high tissue transparency with minimum structural changes and is widely applicable for 3D imaging of various whole biological structures.