Optimizing Tissue Clearing Methods for Improved Imaging of Whole-Mount Retinas

Abstract Gene and cell therapies are promising approaches for restoring vision in hereditary and advanced optic neuropathies. However, these therapeutic approaches must be accurately evaluated through a combination of methods, including advanced imaging, to reach the clinic. We present a whole-mount tissue-clearing methodology to improve imaging of donor neuron integration in the retina following cell transplantation in mice. Mouse retinas were processed using five different clearing methods, comparing tissue transparency, pigmentation, and immunohistochemical clarity. Among the tested methods, ScaleS consistently outperformed its peers, demonstrating a 46% increase in tissue transparency and an 89% increase in immunohistochemical clarity compared to controls. We developed a modified version of ScaleS, termed ScaleH, by adding polyvinyl alcohol to reduce fluorescence decay and enhance sample stability. ScaleH maintained fluorescence stability over extended periods (32% less decay) and proved compatible with immunolabeling and endogenous fluorescent reporters, enabling improved visualization of transplanted human stem cell-derived retinal neurons in the mouse retina. Moreover, ScaleH also improved optic nerve imaging, demonstrating the potential for broader neurobiological applications. Our clearing workflow supports robust, high-resolution imaging for evaluating the integration of transplanted cells in regenerative studies. Competing Interest Statement The authors have declared no competing interest. Footnotes Updated the figures and text throughout