High-Resolution Visualization of Ocular Structures: Clearing Mouse Eye Tissue Using the Free-of-Acrylamide Clearing Technique (FACT)
Helia Noorali1 *
- Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
Abstract: High-resolution visualization of ocular structures and cellular interactions is essential for enhancing our understanding and treatment of eye diseases. Advanced imaging techniques, particularly 3D imaging, allow researchers to capture detailed spatial information of ocular tissues, providing insights into their complex architecture and pathology. This research aims to address the need for improved visualization by utilizing advanced tissue-clearing techniques, specifically the Free-of-Acrylamide Clearing Tissue (FACT) method.
Methods: We initially applied the FACT protocol to eye tissues from C57BL/6 mice, dividing them into five groups: 1) Intact, 2) 10% H2O2 for 3 hours at 55°C, 3) 2% SDS for 48 hours at 37°C, 4) 2% SDS for 72 hours at 37°C, and 5) 4% SDS for 48 hours at 37°C. Retinal morphometry was performed using cryostat sectioning and DAPI staining, and image analysis was conducted via ImageJ. Tubulin protein (TUJ1) was assessed to evaluate protein presence in retinal tissue and the optic nerve. Additionally, SDS-PAGE was performed on samples from all groups to confirm protein preservation. We also utilized GFP-expressing zebrafish to assess the preservation of fluorescent vessels.
Results: The findings revealed a significant decrease in the length of the outer nuclear layer in the 2% SDS-treated group (p < 0.0001). In contrast, no significant morphological changes were noted in the retinal layers treated with 4% SDS for 48 hours at 37°C compared to intact samples. Immunostaining for TUJ1 indicated that protein components remained stable following the clearance process, and SDS-PAGE results were comparable between cleared and intact groups. Importantly, GFP fluorescence was successfully preserved in the samples.
Conclusion: The FACT protocol exhibited varying effects of SDS treatments on ocular tissue morphology. Notably, the 4% SDS group demonstrated minimal adverse effects, suggesting a reduced impact on tissue integrity compared to the 2% SDS treatment. Furthermore, the retention of fluorescence in the mouse retina post-clearing highlights the effectiveness of the FACT protocol in maintaining cellular integrity, thereby enhancing visualization of ocular structures. This technique holds significant promise for 3D imaging applications using light sheet microscopy.
