Preparing Porcine Eyes for Confocal Reflectance Microscopy to Visualize the Vitreous Collagen Fiber Network

The vitreous is a clear gel that fills the space between the lens and retina. Its structure is supported by a network of collagen fibers, which likely influences viscoelastic properties and contributes to retinal disease. However, the organization of this collagen network remains poorly characterized because imaging the vitreous in its native state is challenging. Many imaging techniques applied to the vitreous cause dehydration and distortion, and dye-based methods can introduce artifacts or nonspecific labeling, making it difficult to study the collagen network. In this protocol, we outline: 1) a method for dissecting porcine eyes for imaging; 2) a strategy for mounting the eyes on an inverted microscope; 3) guidelines for imaging the vitreous collagen network in its native state using confocal reflectance microscopy; and 4) instructions for quantitative image analysis of network features. We highlight common pitfalls and emphasize key technical points. We report the application of confocal reflectance microscopy to the vitreous. Compared with prior vitreous imaging strategies, this approach preserves the native structure, orientation, and distribution of vitreous collagen fibers without exogenous dyes or dehydration artifacts. Confocal reflectance microscopy can probe the structural basis of vitreous gel stiffness in health and disease.