<em>En face</em> Cryosectioning of Mouse Retina for High-dimensional Spatial Molecular Analysis

The complex laminar structure of the retina presents significant challenges for comprehensive spatial molecular mapping. Current techniques for investigating tissue architecture and molecular interactions are limited by technical constraints that compromise spatial relationships or restrict molecular profiling. Existing methods like cross-sectioning, whole mount preparations, chemical or mechanical dissociations, and thick en face sections either disrupt tissue integrity, lose critical spatial context, or most often, are simply incompatible with high-dimensional spatial sequencing platforms. This study introduces a cryosectioning technique that produces thin (<20 µm) en face sections while preserving the spatial relationships essential for mapping retinal cell distributions. The method maintains RNA integrity and tissue architecture while generating sections compatible with high-throughput molecular analysis platforms. RNA integrity is maintained because this technique sections fresh frozen tissue, which manufacturers of spatial transcriptomic platforms officially support relative to postfixed tissue. We validate this approach using multiple molecular profiling methods, demonstrating successful integration with both manual RNAscope in situ hybridization (12 targets) and Xenium spatial sequencing (300 targets). The technique consistently preserves tissue architecture across samples while maintaining RNA quality suitable for these sensitive molecular applications. This methodological advancement enables new investigations into retinal cell type distributions, molecular gradients across laminae, and spatial aspects of retinal pathology. The technique's versatility and compatibility with modern spatial biology platforms provide a foundation for comprehensive molecular mapping of the retina's complex cellular organization.