whole mount preparation and confocal imaging of the murine ocular lens anterior region for structural, developmental, and functional insights

Preparation and Imaging of Murine Ocular Lens Whole Mounts

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 organization1,2,3,4,5,6. Surrounding the lens tissue is the capsule, a basement membrane essential for maintaining lens structure and biomechanical properties7,8,9. The lens itself is entirely cellular, consisting of two cell types: epithelial and fiber cells. The epithelial layer consists of a monolayer of cuboidal cells that cover the anterior hemisphere of the lens10. Throughout life, the epithelial cells proliferate and migrate along the lens capsule toward the lens equator. Anterior epithelial cells are quiescent and cobble-stone in cross-section, and near the lens equator, epithelial cells proliferate and start to undergo the differentiation process into new fiber cells11,12. Equatorial epithelial cells transform from randomly packed cells into organized meridional rows with hexagon-shaped cells. Hexagonal cell shape is maintained on the basal side of these differentiating cells while the apical side constricts and anchors at the lens fulcrum or modiolus4,13,14,15. As the equatorial epithelial cells start to elongate into newly formed fiber cells, the apical tips of the cells migrate along the apical surface of anterior epithelial cells toward the anterior pole while the basal tips move along the lens capsule toward the posterior pole. New generations of fiber cells overlay previous generations of cells, creating spherical shells of fibers. During the cell elongation and maturation process, fiber cells substantially alter their morphology11,12,16. These fiber cells form the bulk of the lens mass11,12,16,17,18.
The molecular mechanisms that contribute to establishing intricate lens microstructures, cell morphology, and unique cellular organization are not entirely known. Moreover, the contribution of the lens capsule and cell structure to overall lens function (transparency, lens shape change) is unclear. However, these relationships are being elucidated using new imaging methodology and quantitative assessments of lens structural and cellular features2,4,19,20,21,22. New protocols to image whole lenses that allow for high spatial resolution visualization of the lens capsule, epithelial cells, and peripheral fiber cells have been developed. This includes methodology to quantify capsule thickness, cell size, cell nucleus size and circularity, meridional row order, fiber cell packing, and fiber cell widths. These visualization and image quantification methods allow in-depth morphometric examination and have advantages over other visualization methods (imaging of flat mounts or tissue sections) by preserving overall 3D tissue structure. These methods have permitted for the testing of novel hypotheses and will enable continued advancement in understanding of lens cell pattern development and function.
For the following experiments, we use wild-type and Rosa26-tdTomato mice tandem dimer-Tomato (B6.129(Cg)-Gt(ROSA) (tdTomato)23 (Jackson Laboratories) in the C57BL/6J background between the ages of 6 and 10 weeks, of both sexes. The tdTomato mice allow for visualization of cellular plasma membranes in live lenses via expression of tdTomato protein fused to the N-terminal 8 amino acids of a mutated MARCKS protein that targets the plasma membrane via N-terminal myristylation and internal cysteine-palmitoylation sites23. We also use NMIIAE1841K/E1841K mice24 obtained originally from Dr. Robert Adelstein (National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD). As described previously20, NMIIAE1841K/E1841K&#160;mice in FvBN/129SvEv/C57Bl6 background that has loss of CP49 beaded intermediate filament protein (maintains mature fiber cell morphology and whole lens biomechanics), are backcrossed with C57BL6/J wild-type mice. We screened the offspring for the presence of the wild-type CP49 allele. 
Confocal imaging was performed on a laser-scanning confocal fluorescence microscope with a 20x (NA = 0.8, working distance = 0.55mm, 1x zoom), a 40x (NA = 1.3 oil objective, working distance = 0.2mm, 1x zoom), or a 63x (NA = 1.4 oil objective, working distance = 0.19mm, 1x zoom) magnification. All images were acquired using a pinhole size, which is a determinant of optical section thickness, to 1 Airy Unit (the resultant optical thicknesses are stated in figure legends). Images were processed on Zen Software. Images were exported to .tif format and then imported into FIJI ImageJ Software (imageJ.net).

Author Spotlight: In-Depth Morphometric Examination and Quantification of Native Lens Structure Using Whole Mount Imaging 

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

We aim to determine the molecular mechanisms that establishes the intricate lens architecture and how this established architecture regulates lens function in transparency and lens shape change. To advance the research in our field, we use new imaging methods that allows us to visualize the lens features with high spatial resolution, and this lets us perform quantitative image analysis of lens structures and cellular features. Whole mount imaging is advantageous over visualization of tissue sections or flat mounting procedures as it enables preservation of the overall 3D tissue structure.This allows us to perform in-depth morpho metric examination and quantification on native lens structure. The lens is an integrated biological tissue with specialized functions that rely upon localization and depth dependent geometries of the cells and their associated structures. Using the imaging protocols and quantification methods demonstrated, will allow for a greater understanding of how lens structures and the complex organization of the lens are established.

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Whole Mount Preparation and Confocal Imaging of the Murine Ocular Lens Anterior Region for Structural, Developmental, and Functional Insights  

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Research

JoVE Journal

Biology

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 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 ...