Name: Whole Mount Preparation and Confocal Imaging of the Murine Ocular Lens Anterior Region for Structural, Developmental, and Functional Insights
Uploaded: 2024-01-19T13:50:00
Description: Watch this Scientific Journal Video about Whole Mount Preparation and Confocal Imaging of the Murine Ocular Lens Anterior Region for Structural, Developmental, and Functional Insights at JoVE.com

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

Murine नेत्र लेंस पूरे mounts की तैयारी और इमेजिंग

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).

लेखक स्पॉटलाइट: पूरे माउंट इमेजिंग का उपयोग करके मूल लेंस संरचना की गहराई से मॉर्फोमेट्रिक परीक्षा और मात्रा का ठहराव 

परिधीय लेंस संरचना, सेल आकृति विज्ञान और संगठन की कल्पना और मात्रा निर्धारित करने के लिए पूरे माउंट इमेजिंग

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

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

संरचनात्मक, विकासात्मक और कार्यात्मक अंतर्दृष्टि के लिए मुराइन ओकुलर लेंस पूर्वकाल क्षेत्र की संपूर्ण माउंट तैयारी और कॉन्फोकल इमेजिंग

अगारोज में स्थिरीकरण डिवोट्स बनाकर प्रक्रिया शुरू करें। ऐसा करने के लिए, गर्मी और पीबीएस में 2% agarose मिश्रण, एक माइक्रोवेव का उपयोग कर, जब तक समाधान तरलीकृत है. पिपेट 250 माइक्रोलीटर तरलीकृत एगरोज को एक ग्लास-तल वाले पकवान में।और, एक लचीली प्लास्टिक कवर पर्ची का उपयोग करके, डिश में अगारोज़ को समतल करें। एक बार agarose ठंडा है और पूरी तरह से जम जाता है, कवर पर्ची को हटाने के लिए ठीक टिप संदंश का उपयोग करें. फिर, एक तीन मिलीमीटर बायोप्सी पंच का उपयोग करके, पकवान के केंद्र में agarose में एक छेद बनाएँ.एक नाजुक कार्य पोंछे की मदद से, अतिरिक्त एगरोज को हटा दें। उपयोग करने तक, पीबीएस के साथ हाइड्रेटेड अगारोज मोल्ड को चार डिग्री सेल्सियस पर स्टोर करें। पृथक मिररिंग ओकुलर लेंस को माउंट करने से पहले, लेंस के प्रकार के आधार पर तैयार एगरोज मोल्ड में उपयुक्त माध्यम के दो मिलीलीटर जोड़ें।भ्रूण संदंश का प्रयोग, धीरे agarose में divot में तय या जीवित लेंस हस्तांतरण. फिर, डिश को उल्टे माइक्रोस्कोप स्टेज पर रखें। पुष्टि करें कि लेंस पूर्वकाल क्षेत्र उद्देश्य का सामना करना पड़ के साथ स्थित है, नाभिक धुंधला कल्पना करके.यदि कोई नाभिक नहीं देखा जाता है, तो घुमावदार संदंश का उपयोग धीरे लेंस लगभग 180 डिग्री घुमाएं, ताकि पूर्वकाल क्षेत्र उद्देश्य का सामना करे। अगला, एक confocal माइक्रोस्कोप का उपयोग छवियों को प्राप्त करने के लिए आगे बढ़ें। लेंस कैप्सूल के विज़ुअलाइज़ेशन के लिए, 0.3 माइक्रोन के चरण आकार के साथ, 40x उद्देश्य का उपयोग करके जेड-स्टैक छवियां प्राप्त करें।लेंस कैप्सूल की सतह से पहले पहली छवि प्राप्त करें, जो डब्ल्यूजीए धुंधला द्वारा इंगित की गई है, और उपकला कोशिकाओं की शिखर सतह के बाद अंतिम छवि। उपकला कोशिकाओं कल्पना करने के लिए, 0.3 माइक्रोन के एक कदम आकार के साथ एक 63x उद्देश्य का उपयोग कर जेड स्टैक छवियों का अधिग्रहण.

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Biology

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

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 ...