Name: Preparing Porcine Eyes for Confocal Reflectance Microscopy to Visualize the Vitreous Collagen Fiber Network (Video) | JoVE Journal
Uploaded: 2025-10-17T14:00:00.000+00:00
Duration: 6 min 7 s
Description: Learn confocal reflectance microscopy for visualizing vitreous collagen fiber networks in porcine eyes. This minimal processing method preserves natural structure to study retinal detachment mechanisms.

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5 minutes

National Institutes of Health Core Grant EY014800; Unrestricted Grant from Research to Prevent Blindness, New York, NY, to the Department of Ophthalmology &#38; Visual Sciences, University of Utah; NIH NEI K08 EY034549; Primary Children&#39;s Hospital Foundation; E. Matilda Ziegler Foundation; We thank Drs. Guillaume L. Hoareau and M. Austin Johnson for porcine eyes.

1. Tissue preparation and dissection
NOTE: This research was performed in compliance with institutional guidelines. At our institution, approval was not required from the Institutional Animal Care and Use Committee to perform experiments on ex vivo porcine eyes.
<ol>
	<li>Obtain ex vivo porcine eyes from a meat processing plant. Use the eyes within 48 h of death. 
	NOTE: Although the vitreous appearance does not change with longer postmortem time, the retina and choroid degrade, leading to dispersion of cellular and pigmented debris during dissection. This debris can interfere with fiber imaging and analysis.</li>
	<li>Remove any excess muscle and connective tissue from the globe.</li>
	<li>Place the three-prong pedestal on the base of a cube mold, such as an extra-large silicone ice cube mold (Figure 1A and Figure 2).</li>
	<li>Place the globe on the three-prong pedestal, positioning it so that limbus rests on the prongs.</li>
	<li>Make a 3% agarose gel by adding 3 g of general-purpose agarose to 100 mL of distilled water and then heating the solution until it is dissolved. Once the solution has cooled to around 37 &#176;C, pour it into the mold until the eye is submerged in agarose. 
	NOTE: This will embed the eye in agarose gel to stabilize the eye for dissection.</li>
	<li>Chill the agarose cube for at least 4 h. 
	NOTE: This increases the adhesion of the gel to the eye and will ensure the eye stays immobile during dissection</li>
	<li>After chilling, remove the agarose cube from the mold. Then, remove the pedestal from the cube (Figure 1B).</li>
	<li>Build a three-sided frame in the same dimensions as the agarose cube using a combination of 1 x 8 Lego bricks, plates, and tiles (Figure 3). 
	NOTE: This will secure the cube in place and provide a guide for the blade during dissection. The height of the Lego frame will determine the location the eye will be cut. This can be visualized through the semitransparent agarose gel.</li>
	<li>Place the cooled agarose cube in the Lego frame. Hold a high-profile microtome blade by the blunt edges and align the blade with the top of the Lego frame. Apply gentle downward pressure on the top of the cube and gently drag the microtome blade back and forth through the entire cube and the embedded eye (Figure 1C). 
	NOTE: This will create a section of eye tissue that is embedded in the agarose, which will be referred to as the &#34;eye cap&#34;.</li>
</ol>
2. Mounting sample for imaging
<ol>
	<li>Using the microtome blade, trim approximately 1 mm of agarose from the entire cut surface of the cube (Figure 4A). 
	NOTE: This will leave a small section of eye cap extending above the cut surface of the agarose cube</li>
	<li>Use a tissue wipe or small triangular cellulose sponge to dry the cut surface of the agarose cube, especially near the border of the eye cap.</li>
	<li>Add approximately one teaspoon of all-purpose silicone caulk to a small plastic sandwich bag and cut a 3 mm hole in one of the bottom corners of the bag to create a piping bag for the caulk.</li>
	<li>Using the piping bag, apply a thin bead of caulk to the cut surface of the agarose cube, right along the edge of the eye cap, creating a waterproof border around the sample (Figure 4B). This will keep the vitreous gel from leaking out of the eye cap.</li>
	<li>Place a large coverslip glass over the eye cap and press down so that the cut surface of the eye cap is in contact with the coverslip without any trapped air or fluid (Figure 4C). Confirm that the silicone bead makes a continuous seal with the coverslip. 
	NOTE: If the silicone caulk does not form a continuous seal, vitreous fluid may leak out and allow air into the sample. Exposure to air will cause the vitreous to shrink, resulting in brighter and denser fibers, as well as a liquid area without fibers. If the vitreous is not in contact with the coverslip, it will be difficult to image.</li>
	<li>While holding the agarose cube, invert the sample and place it on the large coverslip adapter for imaging on an inverted microscope (Figure 4D and Figure 5).</li>
</ol>
3. Image acquisition
<ol>
	<li>Use an inverted confocal microscope that is not a spinning disk type. Use a water objective (with water or ocular lubricant gel as a coupler), or oil objective with at least a 0.90 numerical aperture, a magnification between 30x-60x, and a working distance of at least 200 &#181;m.</li>
	<li>Adjust the light path settings for the confocal microscope to obtain reflectance images. In place of the dichroic mirror used to redirect exciting light and transmit emitted light for fluorescence imaging, ensure that there is a beam splitting mirror that redirects a proportion of the illuminating laser and transmits a proportion of the reflected light. 
	NOTE: The reflectance signal from vitreous collagen fibers is somewhat weak, so background noise can impact the ability to image fibers. An improved signal-to-noise ratio was observed with a wavelength of 473 nm, but we were able to image the vitreous collagen fibers with wavelengths ranging from 405 nm to 635 nm.</li>
	<li>Set the detection wavelengths to include the wavelength of the illuminating laser.</li>
	<li>To orient the Z position within the sample, decrease the laser power to the lowest possible setting and identify the flash of reflected light from the coverslip. This will happen at the bottom and the top of the coverslip (Figure 6). 
	NOTE: Near the coverslip, there is a considerable reflective signal in the background. Away from the coverslip, the signal from the reflected light from the collagen fibers decreases.</li>
	<li>Adjust the z position so that it is located above the top of the coverslip and within the sample.</li>
	<li>Increase the laser power to a level higher than usually used for a fluorescence application. 
	NOTE: There is a reflective ring artifact that will be in the center of the imaging field. A combination of zooming in and decentering the imaging field can help exclude this artifact (Figure 7).</li>
	<li>Use line averaging and increase pixel dwell time in the image acquisition settings, which are standard methods for increasing signal-to-noise ratio in confocal microscopes.</li>
</ol>
4. Image analysis of fiber width using CT-FIRE 14
<ol>
	<li>Install CT Fire following the instructions on this website:&#160;https://github.com/uw-loci/curvelets/wiki/Downloads-and-Installation-(CTF). 
	NOTE: The versions used here were &#34;CT-FIRE V3.0 BetaWindows64-2018b&#34; and the MATLAB runtime, &#34;MCR-2018b-win64&#34;. Both must be downloaded for the software to work.</li>
	<li>Convert the images to 8-bit tif and open the 8-bit tif images in CT-FIRE.</li>
	<li>Make sure the software is set to CT-Fire in the top right corner, and then select Run.</li>
	<li>CT-FIRE will automatically upload the results of the analysis to a folder named &#34;ctfireout&#34; located in the same folder as the original image. Examine the quality of extraction by comparing the file starting with the name &#34;OL_ctFIRE&#34; to the original image. To follow this protocol, use the following default settings for image segmentation (i.e., identifying fibers): thresh_im2: 5; s_xlinkbox: 8; thresh_ext: 70; thresh_dang_L: 15; thresh_short_L: 15; s_fiberdir: 4; thresh_linkd: 15; thresh_linka: -150; thresh_flen: 15
	<ol>
		<li>If fibers are not accurately identified, adjust the parameters by clicking the Update button under the parameters section in the top right corner. 
		NOTE: Guidance on how to adjust the parameters can be found on page 20 in section &#34;3 Notes&#34; of the CT-FIRE manual, which is located in the CTFIRE application folder. Additional tips can be found in the article by Liu et al. in section 3.5.215.</li>
	</ol>
	</li>
	<li>Adjust output settings if desired to limit the fibers that appear on the output images. To follow this protocol, use the following default settings: Minimum fiber length [pixels]: 30; Image Res. [dpi]: 300; Fiber line width [0-2]: 0.5; Max fiber width [pixels]: 15</li>
	<li>To convert the units of width from pixel to &#181;m, open the original image in Fiji. Use the dimensions of the image appearing in both pixels and &#181;m in the upper left corner of the display window to calculate a ratio.</li>
</ol>
5. Image analysis of fiber density, curvature, alignment, lacunarity, hyphal growth unit, and fractal dimension using TWOMBLI16
<ol>
	<li>Download TWOMBLI from&#160;https://github.com/wershofe/TWOMBLI.</li>
	<li>Follow the instructions in the file: TwombliDocumentation under &#34;Installation and dependencies&#34;.</li>
	<li>Open the TWOMBLI_v1/programs folder and replace AnamorfProperties.xml file with AnamorfPropertiesEH.xml file (Supplemental File 1).</li>
	<li>In the TWOMBLI_v1/programs folder, replace Twombli_v1.ijm file with Twombli_v2.ijm file (Supplemental File 2).</li>
	<li>Create a folder with empty folders within it called &#34;test HDM,&#34; &#34;test masks,&#34; &#34;HDM,&#34; and &#34;masks&#34;.</li>
	<li>Convert the images to 8-bit tif.</li>
	<li>Put 3-5 images in a &#34;test set input&#34; folder.</li>
	<li>Put all the images to be analyzed in a single folder without subfolders called &#34;eligible&#34;.</li>
	<li>In Fiji, click Plugins | Macros | Run | choose Twombli_v2.ijm in twombli/Programs.</li>
	<li>Follow the prompts to run TWOMBLI. To follow this protocol, use the following parameters: Contrast Saturation, 0.35; Min Line Width, 3; Max Line Width, 3; Min Curvature Window, 40; Max Curvature Window, 40; Minimum Branch Length, 10; Maximum Display HDM, 100; Minimum Gap Diameter, 0 
	NOTE: The parameters for the images may need to be adjusted. The software will guide the user through choosing the parameters, and additional instructions can be found in the file TwombliDocumentation.docx, which came with the program.</li>
</ol>

<ol><li>Sebag, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vitreous+and+vision+degrading+myodesopsia%5BTitle%5D)+AND+%22Prog+Retin+Eye+Res%22%5BJournal%5D)">Vitreous and vision degrading myodesopsia.</a> Prog Retin Eye Res. 79, 100847(2020).</li>
<li>Phillips, J. D., Hwang, E. S., Morgan, D. J., Creveling, C. J., Coats, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+and+mechanics+of+the+vitreoretinal+interface%5BTitle%5D)+AND+%22J+Mech+Behav+Biomed+Mater%22%5BJournal%5D)">Structure and mechanics of the vitreoretinal interface.</a> J Mech Behav Biomed Mater. 134, 105399(2022).</li>
<li>Seider, M. I., Conell, C., Melles, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complications+of+acute+posterior+vitreous+detachment%5BTitle%5D)+AND+%22Ophthalmology%22%5BJournal%5D)">Complications of acute posterior vitreous detachment.</a> Ophthalmology. 129 (1), 67-72 (2022).</li>
<li>Ketkar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vitreomacular+traction+-+a+review%5BTitle%5D)+AND+%22Eye%22%5BJournal%5D)">Vitreomacular traction - a review.</a> Eye. 39 (4), 710-717 (2025).</li>
<li>Filas, B. A., Zhang, Q., Okamoto, R. J., Shui, Y. -B., Beebe, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enzymatic+degradation+identifies+components+responsible+for+the+structural+properties+of+the+vitreous+body%5BTitle%5D)+AND+%22Invest+Ophthalmol+Vis+Sci%22%5BJournal%5D)">Enzymatic degradation identifies components responsible for the structural properties of the vitreous body.</a> Invest Ophthalmol Vis Sci. 55 (1), 55(2014).</li>
<li>Taufalele, P. V., VanderBurgh, J. A., Muñoz, A., Zanotelli, M. R., Reinhart-King, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fiber+alignment+drives+changes+in+architectural+and+mechanical+features+in+collagen+matrices%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Fiber alignment drives changes in architectural and mechanical features in collagen matrices.</a> PLoS One. 14 (5), e0216537(2019).</li>
<li>Bishop, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+macromolecules+and+supramolecular+organisation+of+the+vitreous+gel%5BTitle%5D)+AND+%22Prog+Retin+Eye+Res%22%5BJournal%5D)">Structural macromolecules and supramolecular organisation of the vitreous gel.</a> Prog Retin Eye Res. 19 (3), 323-344 (2000).</li>
<li>Los, L. I., van Luyn, M. J. A., Nieuwenhuis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organization+of+the+rabbit+vitreous+body%3A+Lamellae%2C+Cloquet%27s+channel+and+a+novel+structure%2C+the+%27Alae+canalis+Cloqueti%27%5BTitle%5D)+AND+%22Exp+Eye+Res%22%5BJournal%5D)">Organization of the rabbit vitreous body: Lamellae, Cloquet's channel and a novel structure, the 'Alae canalis Cloqueti'.</a> Exp Eye Res. 69 (3), 343-350 (1999).</li>
<li>Voytik-Harbin, S. L., Brightman, A. O., Waisner, B. Z., Robinson, J. P., Lamar, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small+intestinal+submucosa%3A+A+tissue-derived+extracellular+matrix+that+promotes+tissue-specific+growth+and+differentiation+of+cells+in+vitro%5BTitle%5D)+AND+%22Tissue+Eng%22%5BJournal%5D)">Small intestinal submucosa: A tissue-derived extracellular matrix that promotes tissue-specific growth and differentiation of cells in vitro.</a> Tissue Eng. 4 (2), 157-174 (1998).</li>
<li>Bos, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Collagen+fibril+organisation+in+mammalian+vitreous+by+freeze+etch%2Frotary+shadowing+electron+microscopy%5BTitle%5D)+AND+%22Micron%22%5BJournal%5D)">Collagen fibril organisation in mammalian vitreous by freeze etch/rotary shadowing electron microscopy.</a> Micron. 32 (3), 301-306 (2001).</li>
<li>Bembridge, B. A., Crawford, G. N. C., Pirie, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phase-contrast+microscopy+of+the+animal+vitreous+body%5BTitle%5D)+AND+%22Br+J+Ophthalmol%22%5BJournal%5D)">Phase-contrast microscopy of the animal vitreous body.</a> Br J Ophthalmol. 36 (3), 131-142 (1952).</li>
<li>Filas, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+imaging+of+enzymatic+vitreolysis-induced+fiber+remodeling%5BTitle%5D)+AND+%22Invest+Ophthalmol+Vis+Sci%22%5BJournal%5D)">Quantitative imaging of enzymatic vitreolysis-induced fiber remodeling.</a> Invest Ophthalmol Vis Sci. 55 (12), 8626-8637 (2014).</li>
<li>Los, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+rabbit+as+an+animal+model+for+post-natal+vitreous+matrix+differentiation+and+degeneration%5BTitle%5D)+AND+%22Eye%22%5BJournal%5D)">The rabbit as an animal model for post-natal vitreous matrix differentiation and degeneration.</a> Eye. 22 (10), 1223-1232 (2008).</li>
<li>Bredfeldt, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Computational+segmentation+of+collagen+fibers+from+second-harmonic+generation+images+of+breast+cancer%5BTitle%5D)+AND+%22J+Biomed+Opt%22%5BJournal%5D)">Computational segmentation of collagen fibers from second-harmonic generation images of breast cancer.</a> J Biomed Opt. 19 (1), 016007(2014).</li>
<li>Liu, Y., Keikhosravi, A., Mehta, G. S., Drifka, C. R., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+for+quantifying+fibrillar+collagen+alignment%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Methods for quantifying fibrillar collagen alignment.</a> Methods Mol Biol. 1627, 429-451 (2017).</li>
<li>Wershof, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+FIJI+macro+for+quantifying+pattern+in+extracellular+matrix%5BTitle%5D)+AND+%22Life+Sci+Alliance%22%5BJournal%5D)">A FIJI macro for quantifying pattern in extracellular matrix.</a> Life Sci Alliance. 4 (3), e202000880(2021).</li>
<li>François, J., Victoria-Troncoso, V., Albarran, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+histochemical+structure+of+the+vitreous+fibers+studied+by+phase-contrast+microscopy%5BTitle%5D)+AND+%22Am+J+Ophthalmol%22%5BJournal%5D)">The histochemical structure of the vitreous fibers studied by phase-contrast microscopy.</a> Am J Ophthalmol. 69 (5), 763-773 (1970).</li>
<li>Hwang, E. S., Morgan, D. J., Sun, J., Hartnett, M. E., Toussaint, K. C., Coats, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Confocal+reflectance+microscopy+for+mapping+collagen+fiber+organization+in+the+vitreous+gel+of+the+eye%5BTitle%5D)+AND+%22Biomed+Opt+Express%22%5BJournal%5D)">Confocal reflectance microscopy for mapping collagen fiber organization in the vitreous gel of the eye.</a> Biomed Opt Express. 14 (2), 932(2023).</li>
<li>Harris, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+in+vitro+type+I+collagen+fibrillogenesis+by+transmission+electron+microscopy%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Visualizing in vitro type I collagen fibrillogenesis by transmission electron microscopy.</a> Methods Mol Biol. 1627, 367-383 (2017).</li>
<li>Arevalo, R. C., Urbach, J. S., Blair, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Size-dependent+rheology+of+type+I+collagen+networks%5BTitle%5D)+AND+%22Biophys+J%22%5BJournal%5D)">Size-dependent rheology of type I collagen networks.</a> Biophys J. 99 (8), L65-L67 (2010).</li>
<li>Raub, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Noninvasive+assessment+of+collagen+gel+microstructure+and+mechanics+using+multiphoton+microscopy%5BTitle%5D)+AND+%22Biophys+J%22%5BJournal%5D)">Noninvasive assessment of collagen gel microstructure and mechanics using multiphoton microscopy.</a> Biophys J. 92 (6), 2212-2222 (2007).</li>
</ol>

All authors report no relevant conflicts of interest.

Prior vitreous imaging methods showed artifactual fiber aggregation13, loss of fiber organization, orientation, and location17, or relied on much higher resolution with a smaller field of view, limiting insights into fiber distribution10. Confocal reflectance microscopy of ex vivo, grossly sectioned fresh tissue provides micron-scale images of vitreous collagen fibers while preserving natural fiber organization, orientation, and location. In our hand...

The vitreous is the extracellular matrix between the lens and retina in the eye. Traction from the vitreous on the retina plays a major role in vision loss in diseases such as retinal detachment, macular hole, and vitreomacular traction1,2,3,4. The ability of the vitreous to transmit tractional forces to the retina is determined by its gel stiffness, which in turn depends on the properties of its major components, collagen and hyaluronic acid5. The gel stiffness of the vitreous is likely influenced by features of the network of collagen fibers, since the stiffness of artificial collagen gels is driven by network characteristics such as density, alignment, and fiber width6.
The vitreous collagen fiber network is difficult to image due to high water content and low collagen concentration7, which leads to artifactual distortion during processing for microscopy or transmission electron microscopy. Traditional histology and transmission electron microscopy require processing to harden the vitreous for thin sectioning. The dehydration required for histological embedding causes vitreous fibers to aggregate into a honeycomb configuration8, and ice-crystal formation during freezing increases pore size in the extracellular matrix9. Plastic embedding8 and rapid freezing10 have been applied to the vitreous to minimize artifact, although some residual artifact likely remains. Phase contrast microscopy is a method that does not require sectioning and has been applied to the vitreous, but it requires removing a small piece of vitreous from the eye, thereby losing the original position and orientation of the network11. More recently, quantitative polarized light microscopy has been used to map the average orientation of fibers across a grossly sectioned porcine eye, but individual fibers could not be resolved12. Additionally, dye-based labeling approaches (e.g., trypan blue, picrosirius red, 5-(4,6-Dichlorotriazinyl) Aminofluorescein) can introduce artifacts or nonspecific labeling and were therefore avoided to preserve the native collagen network.
We adopted the sample-preparation method used by Filas et al. for polarized light microscopy study, i.e., gross dissection in an agarose block12, and imaged the specimen using confocal reflectance microscopy, which has not previously been applied to the vitreous. Using this method, we have acquired images of the vitreous collagen fiber network in a hydrated, near-physiological state. This offers several advantages over prior methods, including reduced artifacts compared with freeze-fracture or dehydration-based approaches10,13. This protocol requires an inverted confocal microscope with an immersion objective of at least 30x magnification, a numerical aperture greater than 0.9, and a working distance greater than 200 &#181;m. We include key guidance for sample preparation and image acquisition to facilitate clear resolution of fibers. By maintaining a near-native state of the vitreous collagen matrix, this approach enables researchers to quantitatively analyze fiber orientation, density, and spatial distribution-parameters that are likely crucial to understanding vitreous stiffness and its pathological changes. This technique is particularly suitable for researchers interested in the vitreous collagen network, its structure, and the pathogenesis of vitreoretinal diseases.

<table><tbody><tr><td>Agarose I</td><td>VWR</td><td>0710-500G</td><td></td></tr><tr><td>All-purpose silicone 1 caulk, clear</td><td>GE</td><td>2795576</td><td></td></tr><tr><td>Cellulose sponge (Weck-Cel Eye Spear)</td><td>Beaver -Visitec</td><td>000865</td><td></td></tr><tr><td>Custom 3 prong pedestal&amp;nbsp;</td><td>NA</td><td>NA</td><td></td></tr><tr><td>Custom adapter for mounting large coverslip glass</td><td>NA</td><td>NA</td><td></td></tr><tr><td>Large coverslip glass, 1.42&amp;rdquo; x&amp;nbsp; 2.36&amp;rdquo; no.1</td><td>Ted Pella</td><td>260460-100</td><td></td></tr><tr><td>Lego (classic baseplate, 1 x 8 tiles, 1 x 8 bricks, 1 x 6 plate)</td><td>Lego</td><td>11023, 3008, 3666, 4162</td><td></td></tr><tr><td>Microtome blades (high profile)&amp;nbsp;</td><td>Lieca Biosystems</td><td>14035838926</td><td></td></tr><tr><td>Olympus UPlanSApo 30x silicone objective</td><td>Olympus</td><td>UPLSAPO30XS</td><td></td></tr><tr><td>Silicone large ice cube tray (Samuelworld)</td><td>Amazon</td><td>713243280060</td><td></td></tr><tr><td>Tissue wipes (Kimwipes)&amp;nbsp;</td><td>Kimberly Clark</td><td>34120</td><td></td></tr></tbody></table>

preparing porcine eyes for confocal reflectance microscopy to visualize the vitreous collagen fiber network

Preparing Porcine Eyes for Confocal Reflectance Microscopy to Visualize the Vitreous Collagen Fiber Network

The vitreous is a clear gel that fills the space between the lens and retina. Its structure is supported by a network of collagen fibers, which likely influences&#160;viscoelastic properties and contributes to retinal disease. However, the organization of this collagen network remains poorly characterized because imaging the vitreous in its native state is challenging. Many imaging techniques applied to the vitreous cause dehydration and distortion, and dye-based methods can introduce artifacts or nonspecific labeling, making it difficult to study the collagen network. In this protocol, we outline: 1) a method for dissecting porcine eyes for imaging; 2) a strategy for mounting the eyes on an inverted microscope; 3) guidelines for imaging the vitreous collagen network in its native state using confocal reflectance microscopy; and 4) instructions for quantitative image analysis of network features. We highlight common pitfalls and emphasize key technical points. We report the application of confocal reflectance microscopy to the vitreous. Compared with prior vitreous imaging strategies, this approach preserves the native structure, orientation, and distribution of vitreous collagen fibers without exogenous dyes or dehydration artifacts. Confocal reflectance microscopy can probe the structural basis of vitreous gel stiffness in health and disease.

One eye from each of three young adult pigs (a 4-month-old female, a 5-month-old male, and a 6-month-old female) was imaged after sectioning the eye in an axial plane through the inferior limbus. Six imaging locations were acquired in the X-Y sectioning plane, spaced 4 mm apart. At each imaging location, a contiguous 4 x 4 grid of 70 µm x 70 µm images was obtained (Figure 8). For each pig, each image was analyzed separately for several parameters (Table 1).
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This method visualizes the native structure of collagen fibers in the vitreous body using confocal reflectance microscopy.

The vitreous is a clear gel that fills the space between the lens and retina. Its structure is supported by a network of collagen fibers, which likely ...

Changes in the vitreous lead to retinal detachment. We think that changes in the vitreous collagen fiber network change gel stiffness, causing focal traction on the retina. It's hard to image vitreous.Traditional histology causes shrinkage, loss and a honeycomb artifact since the vitreous is more than 98%water. Most tissues are under 80%water. Our imaging strategy preserves the natural arrangement of collagen fibers in the vitreous since it requires minimal processing, only gross dissection.We hope that this strategy will allow us to discover the root cause of retinal detachment and provide more effective ways to prevent it. To begin, obtain ex vivo porcine eyes from a meat processing plant. Using dissecting scissors or forceps, remove any excess muscle and connective tissue from the globe.Now, position a three-pronged pedestal at the base of a cube mold, such as an extra large silicone ice cube mold, and position the globe on the pedestal, ensuring the limbus rests securely on the prongs. Next, prepare a 3%agarose gel by adding three grams of general purpose agarose to 100 milliliters of distilled water and heat the mixture until fully dissolved. Allow the solution to cool to approximately 37 degrees Celsius and pour it into the mold until the globe is completely submerged.Place the agarose cube in a refrigerator and chill for at least four hours. After chilling, remove the solidified agarose cube from the mold and carefully remove the pedestal from the base of the cube. Construct a three-sided frame matching the dimensions of the agarose cube using one-by-eight LEGO bricks, plates, and tiles.Then place the chilled agarose cube in the LEGO frame. Hold a high-profile microtome blade by the blunt edges and align it with the top of the LEGO frame. Apply gentle downward pressure and drag the blade back and forth through the entire cube and embedded globe.Using a microtome blade, trim approximately one millimeter of agarose from the entire cut surface of the cube. Use a tissue wipe or a small triangular cellulose sponge to gently dry the cut surface of the agarose cube, focusing near the border of the eye cap. Now, add approximately one teaspoon of all-purpose silicone caulk to a small plastic sandwich bag and cut a three millimeter hole in one of the bottom corners of the bag to create a makeshift piping bag.Using the piping bag, apply a thin bead of silicone caulk along the cut surface of the agarose cube, precisely around the edge of the eye cap, to form a waterproof seal. Ensure that the caulk forms a complete and continuous border to prevent vitreous gel leakage. Then place a large cover slip glass over the eye cap and press it down until the surface of the eye cap makes full contact with the glass.Confirm that the silicone caulk forms an unbroken seal with the cover slip. Next, while holding the agarose cube, invert the sample and place it on a large cover slip adapter suitable for imaging on an inverted microscope. Use an inverted confocal microscope that is not a spinning disc type.Select an oil objective for imaging. Adjust the light path settings on the confocal microscope to obtain reflectance images. Replace the dichroic mirror with a beam splitting mirror that redirects part of the illuminating laser and transmits a portion of the reflected light.Then set the detection wavelengths to include the wavelength of the illuminating laser. To orient the Z position within the sample, reduce the laser power to its lowest possible setting. Identify the brief flash of reflected light that appears at both the top and bottom of the cover slip to mark the Z position boundaries.Increase the laser power to a level higher than typically used for fluorescence imaging. Finally, in the image acquisition settings, activate line averaging and increase the pixel dwell time to improve the signal to noise ratio and acquire the images. One eye each from three young adult pigs was imaged after sectioning the eye in an axial plane through the inferior limbus.Representative confocal reflectance images showed visually denser fiber networks in pigs 1 and 2 compared to pig 3. The total length of vitreous fibers per tile was similar across all three pigs, indicating no significant difference in fiber abundance, but the fraction of pixels occupied by signal differed between the three pigs. The curvature of the fibers, which is the mean incremental change in angle along a fiber, and the lacunarity were similar between the three pigs.The hyphal growth unit defined as branch points per fiber length was highest in pig 3, differing significantly from pigs 1 and 2.

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Research

JoVE Journal

Medicine

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