Whole Mount Imaging to Visualize and Quantify Peripheral Lens Structure, Cell Morphology, and Organization

The present protocols describe novel whole mount imaging for the visualization of peripheral structures in the ocular lens with methods for image quantification. These protocols can be used in studies to better understand the relationship between lens microscale structures and lens development/function.

The ocular lens is a transparent flexible tissue that alters its shape to focus light from different distances onto the retina. Aside from a basement membrane surrounding the organ, called the capsule, the lens is entirely cellular consisting of a monolayer of epithelial cells on the anterior hemisphere and a bulk mass of lens fiber cells. Throughout life, epithelial cells proliferate in the germinative zone at the lens equator, and equatorial epithelial cells migrate, elongate, and differentiate into newly formed fiber cells. Equatorial epithelial cells substantially alter morphology from randomly packed cobble-stone-shaped cells into aligned hexagon-shaped cells forming meridional rows. Newly formed lens fiber cells retain the hexagonal cell shape and elongate toward the anterior and posterior poles, forming a new shell of cells that are overlaid onto previous generations of fibers. Little is known about the mechanisms that drive the remarkable morphogenesis of lens epithelial cells to fiber cells. To better understand lens structure, development, and function, new imaging protocols have been developed to image peripheral structures using whole mounts of ocular lenses. Here, methods to quantify capsule thickness, epithelial cell area, cell nuclear area and shape, meridional row cell order and packing, and fiber cell widths are shown. These measurements are essential for elucidating the cellular changes that occur during lifelong lens growth and understanding the changes that occur with age or pathology.

The ocular lens is a flexible, transparent tissue situated at the anterior region of the eye that functions to fine-focus light onto the retina. The ability of the lens to function can be attributed, in part, to its intricate architecture and organization^1,2,3,4,5,6. Surrounding the lens tissue is the capsule, a basement membrane essential for maintaining lens structure and biomechanical properties^7,8,

Mice are housed in the University of Delaware animal facility, maintained in a pathogen-free environment. All animal procedures, including euthanasia by CO₂ inhalation, were conducted in accordance with approved animal protocols by the University of Delaware Institutional Animal Care and Use Committee (IACUC).

1. Whole lens mount preparation and imaging

1. Fixation of lenses for whole mount imaging
   1. Following euthanasia, enucleate eyes and dissec.......

Anterior lens capsule, epithelial cell area, and nuclear area
To analyze lens capsule thickness, we stained lens capsules, in either live or fixed lenses, with WGA. We identified lens epithelial cells by labeling membranes with tdTomato in live lenses (Figure 2A), or via rhodamine-phalloidin staining for F-actin at the cell membranes in fixed lenses (Figure 2B). In an orthogonal (XZ) projection, staining for WGA and tdTomato/rhodamine-phal.......

The protocols described enable high spatial resolution visualization of peripheral lens structures and cells at the anterior and equatorial regions of the lens. In this study, methods for the visualization of lens peripheral structures using intact (live or fixed) lenses where the overall 3D lens architecture is preserved were shown. Additionally, simple methods for morphometric quantitative analysis using publicly available FIJI ImageJ software were provided. The whole mount visualization and quantification methods has .......

This work was supported by the National Eye Institute Grant R01 EY032056 to CC and R01 EY017724 to VMF, as well as the National Institute of General Medical Sciences under grant number P20GM139760. S.T.I was supported by NIH-NIGMS T32-GM133395 as part of the Chemistry-Biology Interface predoctoral training program, and by a University of Delaware Graduate Scholars Award.