Name: Mouse Ocular Lens Equatorial Region Whole Mount Preparation and Confocal Imaging to Understand the Lens Structural-Developmental Relationship
Uploaded: 2024-01-19T13:50:00
Description: Watch this Scientific Journal Video about Mouse Ocular Lens Equatorial Region Whole Mount Preparation and Confocal Imaging to Understand the Lens Structural-Developmental Relationship at JoVE.com

mouse ocular lens equatorial region whole mount preparation and confocal imaging to understand the lens structural-developmental relationship

Préparation et imagerie de montures entières de lentilles oculaires murines

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).

Pleins feux sur l’auteur : Examen morphométrique approfondi et quantification de la structure native de l’objectif à l’aide de l’imagerie à monture entière 

Imagerie à monture entière pour visualiser et quantifier la structure, la morphologie et l’organisation des lentilles périphériques

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.

Watch this Scientific Journal Video about Mouse Ocular Lens Equatorial Region Whole Mount Preparation and Confocal Imaging to Understand the Lens Structural-Developmental Relationship at JoVE.com

Mouse Ocular Lens Equatorial Region Whole Mount Preparation and Confocal Imaging to Understand the Lens Structural-Developmental Relationship  

Lentille oculaire de souris : préparation de la monture entière de la région équatoriale et imagerie confocale pour comprendre la relation structure-développement du cristallin

Commencez toute la préparation de la monture de l’objectif fixe en créant des coins d’agarose d’immobilisation. Pour ce faire, prenez un plat à fond de verre et versez-y environ cinq à six millilitres d’agarose 2% fondue, dans du PBS. Attendez que l’agarose se solidifie.Ensuite, utilisez une lame tranchante pour créer un divot triangulaire dans l’agarose solidifiée. Retirez le quartier d’agarose et ajoutez un millilitre de PBS dans le plat. Aspirer toute agarose résiduelle laissée après la coupe.Créez plusieurs cales dans une seule parabole pour s’adapter à plusieurs lentilles, si nécessaire. Pour conserver le moule, ajoutez-y un millilitre de PBS avant de le maintenir à quatre degrés Celsius. À l’aide d’une pince à épiler incurvée, placez la lentille dans le coin d’agarose contenant un millilitre de PBS.Ajustez la lentille de manière à ce que la région équatoriale soit orientée vers le bas sur la vitre du microscope lorsqu’elle est placée au-dessus de l’objectif confocal. Placez la boîte sur la platine du microscope. Pour confirmer que la région équatoriale est nette, visualisez les noyaux en vous assurant qu’ils sont alignés en rangées.Vérifiez également les cellules épithéliales équatoriales irrégulièrement emballées et de forme, ainsi que les cellules de la rangée méridionale alignées avec précision et de forme hexagonale, indiquées par la coloration de la F-actine au niveau des membranes cellulaires. Un empilement aléatoire des noyaux dans le champ de vision indique que la lentille fait face à l’objectif. Si les noyaux ne peuvent pas être observés, cela indique probablement que la face postérieure du cristallin fait face à l’objectif.Dans de tels cas, utilisez une pince à épiler incurvée pour faire pivoter la lentille jusqu’à ce que les noyaux précisément alignés à l’équateur de la lentille soient observés.

Research

JoVE Journal

Biology

Mouse Ocular Lens Equatorial Region Whole Mount Preparation and Confocal Imaging to Understand the Lens Structural-Developmental Relationship

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

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 ...

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Préparation de la monture complète et imagerie confocale de la région antérieure du cristallin oculaire murin pour des informations structurelles, développementales et fonctionnelles