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.

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
<ol>
	<li>Fixation of lenses for whole mount imaging
	<ol>
		<li>Following euthanasia, enucleate eyes and dissec...

Preparation and Imaging of Murine Ocular Lens Whole Mounts

<ol>
	<li>Gokhin, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tmod1+and+CP49+synergize+to+control+the+fiber+cell+geometry,+transparency,+and+mechanical+stiffness+of+the+mouse+lens.">Tmod1 and CP49 synergize to control the fiber cell geometry, transparency, and mechanical stiffness of the mouse lens.</a> PLoS One. 7 (11), e48734 (2012).</li><li>Cheng, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+in+eye+lens+biomechanics,+morphology,+refractive+index+and+transparency.">Age-related changes in eye lens biomechanics, morphology, refractive index and transparency.</a> Aging (Albany NY). 11 (24), 12497-12531 (2019).</li><li>Cheng, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tropomodulin+1+regulation+of+actin+is+required+for+the+formation+of+large+paddle+protrusions+between+mature+lens+fiber+cells.">Tropomodulin 1 regulation of actin is required for the formation of large paddle protrusions between mature lens fiber cells.</a> Invest Ophthalmol Vis Sci. 57 (10), 4084-4099 (2016).</li><li>Parreno, J., Cheng, C., Nowak, R. B., Fowler, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+mechanical+strain+on+mouse+eye+lens+capsule+and+cellular+microstructure.">The effects of mechanical strain on mouse eye lens capsule and cellular microstructure.</a> Mol Biol Cell. 29 (16), 1963-1974 (2018).</li><li>Sindhu Kumari, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Aquaporin+0+in+lens+biomechanics.">Role of Aquaporin 0 in lens biomechanics.</a> Biochem Biophys Res Commun. 462 (4), 339-345 (2015).</li><li>Martin, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arvcf+dependent+adherens+junction+stability+is+required+to+prevent+age-related+cortical+cataracts.">Arvcf dependent adherens junction stability is required to prevent age-related cortical cataracts.</a> Front Cell Dev Biol. 10, 840129 (2022).</li><li>Danysh, B. P., Duncan, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lens+capsule.">The lens capsule.</a> Exp Eye Res. 88 (2), 151-164 (2009).</li><li>Mekonnen, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lens+capsule+significantly+affects+the+viscoelastic+properties+of+the+lens+as+quantified+by+optical+coherence+elastography.">The lens capsule significantly affects the viscoelastic properties of the lens as quantified by optical coherence elastography.</a> Front Bioeng Biotechnol. 11, 1134086 (2023).</li><li>Fincham, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+function+of+the+lens+capsule+in+the+accommodation+of+the+eye.">The function of the lens capsule in the accommodation of the eye.</a> Trans Optical Society. 30 (3), 101 (1929).</li><li>Cheng, C., Nowak, R. B., Fowler, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lens+actin+filament+cytoskeleton:+Diverse+structures+for+complex+functions.">The lens actin filament cytoskeleton: Diverse structures for complex functions.</a> Exp Eye Res. 156, 58-71 (2017).</li><li>Bassnett, S., Sikic, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lens+growth+process.">The lens growth process.</a> Prog Retin Eye Res. 60, 181-200 (2017).</li><li>Sikic, H., Shi, Y., Lubura, S., Bassnett, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+full+lifespan+model+of+vertebrate+lens+growth.">A full lifespan model of vertebrate lens growth.</a> R Soc Open Sci. 4 (1), 160695 (2017).</li><li>Cheng, C., Ansari, M. M., Cooper, J. A., Gong, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EphA2+and+Src+regulate+equatorial+cell+morphogenesis+during+lens+development.">EphA2 and Src regulate equatorial cell morphogenesis during lens development.</a> Development. 140 (20), 4237-4245 (2013).</li><li>Sugiyama, Y., Akimoto, K., Robinson, M. L., Ohno, S., Quinlan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell+polarity+protein+aPKClambda+is+required+for+eye+lens+formation+and+growth.">A cell polarity protein aPKClambda is required for eye lens formation and growth.</a> Dev Biol. 336 (2), 246-256 (2009).</li><li>Zampighi, G. A., Eskandari, S., Kreman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+organization+of+the+mammalian+lens.">Epithelial organization of the mammalian lens.</a> Exp Eye Res. 71 (4), 415-435 (2000).</li><li>Lovicu, F. J., Robinson, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Development of the Ocular Lens. , (2011).</li><li>Kuszak, J. R., Zoltoski, R. K., Sivertson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibre+cell+organization+in+crystalline+lenses.">Fibre cell organization in crystalline lenses.</a> Exp Eye Res. 78 (3), 673-687 (2004).</li><li>Cvekl, A., Ashery-Padan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+and+molecular+mechanisms+of+vertebrate+lens+development.">The cellular and molecular mechanisms of vertebrate lens development.</a> Development. 141 (23), 4432-4447 (2014).</li><li>Vu, M. P., Cheng, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+immunofluorescence+staining+of+bundles+and+single+fiber+cells+from+the+cortex+and+nucleus+of+the+eye+lens.">Preparation and immunofluorescence staining of bundles and single fiber cells from the cortex and nucleus of the eye lens.</a> J Vis Exp. (196), e65638 (2023).</li><li>Islam, S. T., Cheng, C., Parreno, J., Fowler, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonmuscle+myosin+IIA+regulates+the+precise+alignment+of+hexagonal+eye+lens+epithelial+cells+during+fiber+cell+formation+and+differentiation.">Nonmuscle myosin IIA regulates the precise alignment of hexagonal eye lens epithelial cells during fiber cell formation and differentiation.</a> Invest Ophthalmol Vis Sci. 64 (4), 20 (2023).</li><li>Patel, S. D., Aryal, S., Mennetti, L. P., Parreno, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+mount+staining+of+lenses+for+visualization+of+lens+epithelial+cell+proteins.">Whole mount staining of lenses for visualization of lens epithelial cell proteins.</a> MethodsX. 8, 101376 (2021).</li><li>Parreno, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodologies+to+unlock+the+molecular+expression+and+cellular+structure+of+ocular+lens+epithelial+cells.">Methodologies to unlock the molecular expression and cellular structure of ocular lens epithelial cells.</a> Front Cell Dev Biol. 10, 983178 (2022).</li><li>Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+global+double-fluorescent+Cre+reporter+mouse.">A global double-fluorescent Cre reporter mouse.</a> Genesis. 45 (9), 593-605 (2007).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+MYH9-related+disease:+mutations+in+nonmuscle+myosin+II-A.">Mouse models of MYH9-related disease: mutations in nonmuscle myosin II-A.</a> Blood. 119 (1), 238-250 (2012).</li><li>Cheng, C., Gokhin, D. S., Nowak, R. B., Fowler, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+application+of+glass+coverslips+to+assess+the+compressive+stiffness+of+the+mouse+lens:+Strain+and+morphometric+analyses.">Sequential application of glass coverslips to assess the compressive stiffness of the mouse lens: Strain and morphometric analyses.</a> J Vis Exp. (111), e53986 (2016).</li><li>Riedl, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifeact:+a+versatile+marker+to+visualize+F-actin.">Lifeact: a versatile marker to visualize F-actin.</a> Nat Methods. 5 (7), 605-607 (2008).</li><li>Lukinavicius, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorogenic+probes+for+live-cell+imaging+of+the+cytoskeleton.">Fluorogenic probes for live-cell imaging of the cytoskeleton.</a> Nat Methods. 11 (7), 731-733 (2014).</li></ol>

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 ...

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).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3 mm Biopsy Punch</td>
			<td>Acuderm Inc</td>
			<td>NC9084780</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Apex BioResearch Products</td>
			<td>20-102GP</td>
			<td></td>
		</tr>
		<tr>
			<td>Antimycotic/Antibiotic</td>
			<td>Cytiva</td>
			<td>SV30079.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (Fraction V)</td>
			<td>Prometheus</td>
			<td>25-529</td>
			<td></td>
		</tr>
		<tr>
			<td>Delicate task wipes</td>
			<td>Kimwipe</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottomed dish (Fluorodish)</td>
			<td>World Precision International</td>
			<td>FD35-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoescht 33342</td>
			<td>Biotium</td>
			<td>40046</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser scanning confocal Microscope 880</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MatTek Imaging Dish</td>
			<td>MatTek Life Sciences</td>
			<td>P35G-1.5-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>100503-917</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>GenClone</td>
			<td>25-507B</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red-free medium 199</td>
			<td>Gibco</td>
			<td>11043023</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine-Phalloidin</td>
			<td>Thermo Fisher</td>
			<td>00027</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X100</td>
			<td>Sigma-Aldrich</td>
			<td>11332481001</td>
			<td></td>
		</tr>
		<tr>
			<td>WGA-640</td>
			<td>Biotium</td>
			<td>CF 640R</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 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.

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.

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...

Watch this Scientific Journal Video about In-Depth Morphometric Examination and Quantification of Native Lens Structure Using Whole Mount Imaging at JoVE.com

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 ...

whole-mount-imaging-to-visualize-quantify-peripheral-lens-structure

Research

JoVE Journal

Biology

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

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

693 Views.  University of Delaware.  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.

Preparation and Imaging of Murine Ocular Lens Whole Mounts

Summary