Making eyes invisible to make things more visible: An optimized tissue clearing approach for three-dimensional imaging and high-throughput analysis of whole rodent eyes

Abstract

Purpose : The mouse eye serves as a useful tool to model a variety of pathologies and processes related to development, neurological disorders, regenerative therapies, vascular angiogenesis and tumorigenesis. Light sheet fluorescence microscopy allows for high-throughput analysis of optically cleared tissue samples, however established clearing methods do not address challenges posed by tissues such as the eye, which contains pigments that mitigate imaging accessibility. Thus, we have developed an optimized tissue clearing protocol for the rodent eye and have applied it to the analysis of three-dimensionally relevant features.

Methods : Mouse eyes were dissected, permeabilized, fixed and depigmented prior to clearing using an optimized version of the CUBIC clearing protocol. Imaging using the LaVision UltraMicroscope II light sheet, and analysis with Imaris 9.2.0 was performed on photoreceptor transplanted, retinoblastoma tumor, and Norrie disease eyes.

Results : We demonstrate using our optimized protocol that pigmented rodent eyes can undergo tissue clearing and subsequent high-throughput analysis using a light sheet fluorescent microscope. We demonstrate that our method provides single-cell resolution of retinal cells and allows for rapid confirmation of a successful sub-retinal injection and analysis of the topographic distribution of deposited cells. We have also applied our method for high-throughput analysis of the 3D topographical distribution and volume of retinoblastoma tumors. The 3D volumetric analysis of the inner retinal vasculature and choroidal vasculature may also be determined and compared to models with vasculature defects.

Conclusions : Our tissue clearing, depigmentation and staining protocol is a more efficient and effective method, than traditional sectioning methods, to study retinal transplantation, tumorigenesis, vasculature and other processes in the eye. This method of analysis may also provide new information, previously not attainable due to artifacts or limitations present in tissue processing.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.