Optimizing tissue clearing methods for improved imaging of whole-mount retinas and optic nerves.

BACKGROUND

Gene and cell therapies hold promise for restoring vision in hereditary and advanced optic neuropathies. Accurate evaluation of these therapies requires advanced imaging methods that can visualize transplanted cells within intact retinal tissue.

NEW METHOD: We present a whole-mount tissue-clearing workflow optimized for the mouse retina and optic nerve to improve visualization of donor neuron integration following cell transplantation. Five clearing methods were evaluated, and a modified protocol, ScaleH, was developed by adding polyvinyl alcohol to ScaleS to improve fluorescence preservation.

RESULTS

ScaleS yielded the highest transparency (46 % increase) and immunohistochemical clarity (89 % increase) in the retina among the tested methods. ScaleH retained comparable clarity while significantly reducing fluorescence decay over time (32 % less decay). ScaleH was compatible with endogenous reporters and immunolabeling, enabling detailed imaging of transplanted human stem cell-derived retinal neurons in the retinal ganglion cell layer, as well as visualization of neurites, microglia, and cell nuclei in the optic nerve.

COMPARISON WITH EXISTING

METHODS

Compared to other clearing protocols, ScaleH provided superior fluorescence retention and stability without compromising optical clarity. Its compatibility with both immunostaining and endogenous fluorescent proteins supports broad application in imaging.

CONCLUSIONS

ScaleH is a reliable and high-resolution clearing method for imaging whole-mount retinas and optic nerves. It facilitates robust assessment of donor cell integration in regenerative ophthalmology and may be broadly applicable in neurobiological research.