No funding was received for this manuscript.

All procedures performed in the following protocol involving human participants were in accordance with the ethical standards of the institutional research committee of the University of Cologne and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
1. Prosthetic eye customization
<ol>
	<li>Select one of the &#34;half-done&#34; cryolite glass eyes based on the best matching iris color to the healthy fellow eye of the patient (Figure 3).</li>
	<li>Examine the fitting of the current prosthetic eye. To do so, let the patient look straight ahead. Pay special attention to the retention of the prosthesis, the viewing direction, the eye lid contour (ptosis, entropion, and ectropion), as well as to the size and volume (exophthalmos and enophthalmos) of the current prosthesis.</li>
	<li>Remove the current prosthetic eye with help of a contact lens suction cup for hard contact lenses.</li>
	<li>Examine the anophthalmic eye socket without the prosthesis and pay attention to a potential inflammation of conjunctiva, the volume filling of the orbital implant, if the orbital implant is visible through the conjunctiva, and if the fornices and sulci are deep enough for a good fitting prosthesis. If there are any major concerns regarding one of these points, an examination by an ophthalmic surgeon should be performed before manufacturing a new prosthesis.</li>
	<li>Take the selected &#34;half-done&#34; cryolite glass eye with the ocularist forceps and in the other hand take a hollow skewer that will be used later as a mouthpiece for blowing the glass prosthesis. Heat both slowly to 600 &#176;C with a Bunsen burner while continuously rotating it, and melt the skewer at the open end of the &#34;half-done&#34; cryolite glass eye. Open the forceps and lay it down.</li>
	<li>Heat the &#34;half-done&#34; cryolite glass eye continuously (Figure 4). Using the healthy fellow eye as a model for the color, shape, and quantity of the conjunctival vessels, draw the vessels on the white sclera with heated glass stems in different colors (mostly red, brown, or yellow) (Figure 5).</li>
	<li>Heat the whole &#34;half-done&#34; cryolite glass eye while continuously rotating it so that the drawn vessels merge with the white cryolite glass and to produce a very smooth surface.</li>
	<li>Modify the shape and the volume of the of the cryolite glass prosthetic eye by suction and blowing in the mouthpiece. Keep rotating the glass eye in the flame of the Bunsen burner from time to time. Use the old prosthesis as a template for this step, but if necessary, modify the shape and the volume of the new prosthesis based on the findings of the previous examinations.</li>
	<li>Heat a transparent glass stem and melt it at the pupil of the cryolite glass prosthetic eye while continuously rotating the glass eye (Figure 6).</li>
	<li>While continuously rotating the &#34;half-done&#34; glass prosthetic eye, melt the glass at the rear of the prosthesis (Figure 6 and Figure 7) and reduce the volume of the rear by suction with help of the mouthpiece so that the back side shape is nearly equal to the sample prosthesis or the desired shape.</li>
	<li>Melt the glass stem at the front side away and heat the front side of the prosthesis again to produce a very smooth surface (Figure 8).</li>
	<li>Take the front side of the prosthesis with the forceps again, form the final shape of the back side with help of the skewer (Figure 9), and then melt the skewer away (Figure 10).</li>
	<li>Heat the whole prosthesis for fire polishing again, especially at the back side and rotate the prosthesis until the surface is very smooth all over.</li>
	<li>Put the prosthesis in a preheated metal container and let it slowly cool down (Figure 11).</li>
	<li>Insert the prosthesis and check the fitting as described in step 1.2 (Figure 12).</li>
	<li>If necessary, modify the shape of the prosthesis again (repeat steps 1.8&#8211;1.15).</li>
</ol>

<ol>
	<li>Hintschich, C., Baldeschi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rehabilitation+of+anophthalmic+patients.+Results+of+a+survey.">Rehabilitation of anophthalmic patients. Results of a survey.</a> Ophthalmologe. 98 (1), 74-80 (2001).</li><li>Rokohl, A. C., Mor, J. M., Trester, M., Koch, K. R., Heindl, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rehabilitation+of+Anophthalmic+Patients+with+Prosthetic+Eyes+in+Germany+Today+-+Supply+Possibilities,+Daily+Use,+Complications+and+Psychological+Aspects.">Rehabilitation of Anophthalmic Patients with Prosthetic Eyes in Germany Today - Supply Possibilities, Daily Use, Complications and Psychological Aspects.</a> Klinische Monatsbl&#228;tter Augenheilkunde. 236 (1), 54-62 (2019).</li><li>Rokohl, A. C., Koch, K. R., Trester, M., Heindl, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryolite+glass+ocular+prostheses+and+coralline+hydroxyapatite+implants+for+eye+replacement+following+enucleation.">Cryolite glass ocular prostheses and coralline hydroxyapatite implants for eye replacement following enucleation.</a> Ophthalmologe. 115 (9), 793-794 (2018).</li><li>Rokohl, A. 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=Concerns+of+anophthalmic+patients-a+comparison+between+cryolite+glass+and+polymethyl+methacrylate+prosthetic+eye+wearers.">Concerns of anophthalmic patients-a comparison between cryolite glass and polymethyl methacrylate prosthetic eye wearers.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 256 (6), 1203-1208 (2018).</li><li>Rokohl, A. C., Trester, M., Pine, K. R., Heindl, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+of+breakage+of+cryolite+glass+prosthetic+eyes.">Risk of breakage of cryolite glass prosthetic eyes.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 257 (2), 437-438 (2019).</li><li>den Tonkelaar, I., Henkes, H. E., van Leersum, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herman+Snellen+(1834-1908)+and+Muller's+'reform-auge'.+A+short+history+of+the+artificial+eye.">Herman Snellen (1834-1908) and Muller's 'reform-auge'. A short history of the artificial eye.</a> Documenta Ophthalmologica. 77 (4), 349-354 (1991).</li><li>Koch, K. R., 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=Ocular+prosthetics.+Fitting,+daily+use+and+complications.">Ocular prosthetics. Fitting, daily use and complications.</a> Ophthalmologe. 113 (2), 133-142 (2016).</li><li>Pine, K. R., Sloan, B. H., Jacobs, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Clinical ocular prosthetics. 1st ed. , (2015).</li><li>Buckel, M., Bovet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+eye+as+an+art+form:+the+ocular+prosthesis.">The eye as an art form: the ocular prosthesis.</a> Klinische Monatsbl&#228;tter Augenheilkunde. 200 (5), 594-595 (1992).</li><li>Rokohl, A. 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=Concerns+of+Anophthalmic+Patients+Wearing+Cryolite+Glass+Prosthetic+Eyes.">Concerns of Anophthalmic Patients Wearing Cryolite Glass Prosthetic Eyes.</a> Ophthalmic Plastic and Reconstructive Surgery. 34 (4), 369-374 (2018).</li><li>Rokohl, A. 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=Cryolite+glass+prosthetic+eyes-the+response+of+the+anophthalmic+socket.">Cryolite glass prosthetic eyes-the response of the anophthalmic socket.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. , (2019).</li><li>Thiesmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motility+and+lid+changes+with+coralline+hydroxyapatite+orbital+implants+and+cryolite+glass+ocular+prostheses.">Motility and lid changes with coralline hydroxyapatite orbital implants and cryolite glass ocular prostheses.</a> Ophthalmologe. 115 (9), 794-796 (2018).</li><li>Thiesmann, R., Anagnostopoulos, A., Stemplewitz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+results+of+the+compatibility+of+a+coralline+hydroxyapatite+implant+as+eye+replacement.">Long-term results of the compatibility of a coralline hydroxyapatite implant as eye replacement.</a> Ophthalmologe. 115 (2), 131-136 (2018).</li><li>Chin, K., Margolin, C. B., Finger, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+ocular+prosthesis+insertion+improves+quality+of+life+after+enucleation.">Early ocular prosthesis insertion improves quality of life after enucleation.</a> Optometry. 77 (2), 71-75 (2006).</li><li>Pine, K., Sloan, B., Stewart, J., Jacobs, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concerns+of+anophthalmic+patients+wearing+artificial+eyes.">Concerns of anophthalmic patients wearing artificial eyes.</a> Clinical and Experimental Ophthalmology. 39 (1), 47-52 (2011).</li><li>Pine, K. R., Sloan, B., Jacobs, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosocial+profile+of+New+Zealand+prosthetic+eye+wearers.">Biosocial profile of New Zealand prosthetic eye wearers.</a> New Zealand Medical Journal. 125 (1363), 29-38 (2012).</li><li>Pine, K. R., Sloan, B., Stewart, J., Jacobs, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+response+of+the+anophthalmic+socket+to+prosthetic+eye+wear.">The response of the anophthalmic socket to prosthetic eye wear.</a> Clinical and Experimental Optometry. 96 (4), 388-393 (2013).</li><li>Pine, K. R., Sloan, B. H., Jacobs, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+proposed+model+of+the+response+of+the+anophthalmic+socket+to+prosthetic+eye+wear+and+its+application+to+the+management+of+mucoid+discharge.">A proposed model of the response of the anophthalmic socket to prosthetic eye wear and its application to the management of mucoid discharge.</a> Medical Hypotheses. 81 (2), 300-305 (2013).</li><li>Pine, N. S., de Terte, I., Pine, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+investigation+into+discharge,+visual+perception,+and+appearance+concerns+of+prosthetic+eye+wearers.">An investigation into discharge, visual perception, and appearance concerns of prosthetic eye wearers.</a> Orbit. 36 (6), 401-406 (2017).</li><li>Pine, K. R., Sloan, B., Jacobs, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+measurement+tools+for+prosthetic+eye+research.">The development of measurement tools for prosthetic eye research.</a> Clinical and Experimental Optometry. 96 (1), 32-38 (2013).</li><li>Pine, K. R., Sloan, B., Jacobs, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deposit+buildup+on+prosthetic+eyes+and+implications+for+conjunctival+inflammation+and+mucoid+discharge.">Deposit buildup on prosthetic eyes and implications for conjunctival inflammation and mucoid discharge.</a> Clinical Ophthalmology. 6, 1755-1762 (2012).</li><li>Pine, K., Sloan, B., Stewart, J., Jacobs, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+prosthetic+eye+wearers+to+investigate+mucoid+discharge.">A survey of prosthetic eye wearers to investigate mucoid discharge.</a> Clinical Ophthalmology. 6, 707-713 (2012).</li><li>H&#228;rting, F., Fl&#246;rke, O. W., Bornfeld, N., Trester, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+changes+in+glass+eye+prostheses.">Surface changes in glass eye prostheses.</a> Klinische Monatsbl&#228;tter Augenheilkunde. 185 (4), 272-275 (1984).</li><li>Worrell, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hollow+Prosthetic+Eyes.">Hollow Prosthetic Eyes.</a> Ophthalmic Plastic and Reconstructive Surgery. 32 (6), 132-135 (2016).</li><li>Minoura, K., 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=Antibacterial+effects+of+the+artificial+surface+of+nanoimprinted+moth-eye+film.">Antibacterial effects of the artificial surface of nanoimprinted moth-eye film.</a> PLoS One. 12 (9), 0185366 (2017).</li><li>Litwin, A. S., Worrell, E., Roos, J. C., Edwards, B., Malhotra, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+We+Improve+the+Tolerance+of+an+Ocular+Prosthesis+by+Enhancing+Its+Surface+Finish.">Can We Improve the Tolerance of an Ocular Prosthesis by Enhancing Its Surface Finish.</a> Ophthalmic Plastic and Reconstructive Surgery. , (2017).</li><li>Kavlekar, A. A., Aras, M. A., Chitre, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+innovative+and+simple+approach+to+fabricate+a+hollow+ocular+prosthesis+with+functional+lubricant+reservoir:+A+solution+to+artificial+eye+comfort.">An innovative and simple approach to fabricate a hollow ocular prosthesis with functional lubricant reservoir: A solution to artificial eye comfort.</a> The Journal of the Indian Prosthodontic Society. 17 (2), 196-202 (2017).</li></ol>

Alexander C. Rokohl, Joel M. Mor, Niklas Loreck, Konrad R. Koch, and Ludwig M. Heindl have no financial or proprietary interest in any material or method mentioned in the article. The participant in this study was recruited from the Trester-Institute for Ocular Prosthetics and Artificial Eyes in Cologne that is owned and operated by Marc Trester.&#160;

Following enucleation with an orbital implant, a conformer has to be inserted for two weeks (Figure 1) in order to prevent scarring of the conjunctival fornices and subsequent inserting of a prosthesis2,3,4,7,12,13. Because an early ocular prosthesis insertion improves quality of life after enucl...

The purpose of the present manuscript is to comprehensively demonstrate the technique of manufacturing a customized cryolite glass prosthetic that is long forgotten outside the German-speaking countries (Figure 1). This manuscript also focuses on major advantages of this technique. These include a very smooth surface of the prosthesis due to fire polishing, the light weight of the prosthesis due to the hollow design, high levels of patient satisfaction, and the need of only one appointment for manufacturing of the customized prosthesis1,2,3,4,5. This article also gives ophthalmologists better insights into ocularistic care in order to improve essential interprofessional collaboration1,2,3,4,5.
In 1832, the glassblower Ludwig Uri M&#252;ller from Thuringia, Germany, developed the cryolite glass prosthetic eye based on the class-leading models made in France4. Benefits of cryolite glass included a better look, better tolerability, easier processing, and longer durability than previous glass eyes4,6,7,8. Herman Snellen, a Dutch eye surgeon, used this cryolite glass to produce a lightweight hollow prosthetic eye in 18804,6,7,8. This lightweight prosthetic eye, the Snellen &#8216;reform eye&#8217;, increased the volume of prosthetic eyes, resulting in better fitting into larger eye sockets following the introduction of enucleation procedures made possible by the development of anesthesia and asepsis4,8. Twenty years later, cryolite glass had become the most commonly used material for prosthetic eyes. Germany developed into the manufacturing center of prosthetic eyes globally2,4,5,7,8. At the start of the second world war, German cryolite glass eyes became unavailable outside of the German-speaking area. Therefore, (poly)methyl methacrylate (PMMA) became a substitute material for prosthetic eyes4,7,8, and today PMMA is the most commonly used material for prosthetic eyes globally4,5,8. Notwithstanding, in German speaking countries, over 90% of ocularists still manufacture customized prostheses using the cryolite glass from Thuringia2,3,4,5,7,8,9,10,11,12,13. Each customized cryolite glass prosthetic eye is produced in two major steps: the first step is to produce a &#34;half-done&#34; cryolite glass eye that conforms to a white sphere with an iris and a pupil (Figure 2). The second and decisive step is to customize the &#34;half-done&#34; cryolite glass prosthetic eye for the respective patient. To that end, a &#34;half-done&#34; cryolite glass eye is selected from thousands (Figure 3) based on the best matching iris color to the patient&#39;s healthy fellow eye.
The following protocol presents customizing a selected &#34;half-done&#34; cryolite glass eye for a specific patient. This step lasts about 25&#8211;35 min.

<table><tbody><tr><td>Bunsen burner with gas and air flow over a fire-resistant worktop made from anodised stainless steel</td><td></td><td></td><td></td></tr><tr><td>Hollow skewer</td><td></td><td></td><td></td></tr><tr><td>Ocularist forceps</td><td></td><td></td><td></td></tr><tr><td>Preheated metal container to 500 degree celsius</td><td></td><td></td><td></td></tr><tr><td>Pre-produced &quot;half-done&quot; cryolite glass eye</td><td></td><td></td><td></td></tr><tr><td>Transparent glass stem</td><td></td><td></td><td></td></tr><tr><td>Various preproduced glass stems in different colors</td><td></td><td></td><td></td></tr></tbody></table>

customizing a cryolite glass prosthetic eye

In Germany, Austria, and Switzerland, over 90% of ocularists still manufacture customized prostheses using cryolite glass from Thuringia. The present manuscript demonstrates this long-forgotten technique in detail. This manuscript shows some major advantages of manufacturing prosthetic eyes using cryolite glass in comparison to poly(methyl methacrylate) (PMMA). These advantages include a lighter weight of the prosthesis, higher levels of patient satisfaction, and only one appointment necessary for the customized manufacturing. Potential risk of breakage seems not to be a critical disadvantage for glass prosthetic eye wearers. However, in some patients, manufacturing a well-fitting prosthetic eye is not possible or reasonable due to anophthalmic socket complications such as post nucleation socket syndrome, scarred fornices, or an orbital implant exposure. This article gives ophthalmologists a better insight into ocularistic care in order to improve the essential interprofessional collaboration between ocularists and ophthalmologists.

Optimal results include a new prosthetic cryolite glass eye that fits very well, is comfortable, has a good motility, and the appearance with the prosthetic eye, including the eye lid contour, is nearly symmetrical to the healthy fellow eye (Figure 12).
Suboptimal results can result if the new prosthetic cryolite glass eye fits and is comfortable, but there are concerns regarding the cosmetic results. If a prosthesis does not fit perfectly, the appearance, includi...

Watch this Scientific Journal Video about Customizing a Cryolite Glass Prosthetic Eye at JoVE.com

Customizing a Cryolite Glass Prosthetic Eye

This manuscript shows each step of customizing a cryolite glass prosthetic eye including some major advantages of the use of cryolite glass for manufacturing an eye prosthesis compared to poly(methyl methacrylate). In addition, this manuscript gives ophthalmologists better insight into the ocularistic care that could improve interprofessional collaboration.

In Germany, Austria, and Switzerland, over 90% of ocularists still manufacture customized prostheses using cryolite glass from Thuringia. The present ...

customizing-a-cryolite-glass-prosthetic-eye

Research

JoVE Journal

Medicine

10.5K Views.  Faculty of Medicine and University Hospital Cologne. PMMA is momenteel het meest gebruikte materiaal voor protheseogen wereldwijd. Echter, in Duitsland, Oostenrijk en Zwitserland, meer dan 90% van de ocularisten zijn nog steeds de productie van aangepaste prothesen met behulp van cryolite glas van Turnia Deze video zal aantonen elke stap van het aanpassen van een cryolite glazen oog in detail, en zal oogartsen betere inzichten in ocularistische zorg te geven. Deze inzichten zullen helpen om de essentiële en professionele samenwerking tussen oc...

Video: Customizing a Cryolite Glass Prosthetic Eye

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are
 preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of
 data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative 
 cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the 
 assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This 
 cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM 
 studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols 
 for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a 
 reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

Bioengineering

Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis

With the recent development of retinal prostheses, it is important to develop reliable techniques for assessing the safety of these devices in preclinical studies. However, the standard fixation, preparation, and automated histology procedures are not ideal. Here we describe new procedures for evaluating the health of the retina directly adjacent to an implant. Retinal prostheses feature electrode arrays in contact with eye tissue. Previous methods have not been able to spatially localize the ocular tissue adjacent to individual electrodes within the array. In addition, standard histological processing often results in gross artifactual detachment of the retinal layers when assessing implanted eyes. Consequently, it has been difficult to assess localized damage, if present, caused by implantation and stimulation of an implanted electrode array. Therefore, we developed a method for identifying and localizing the ocular tissue adjacent to implanted electrodes using a (color-coded) dye marking scheme, and we modified an eye fixation technique to minimize artifactual retinal detachment. This method also rendered the sclera translucent, enabling localization of individual electrodes and specific parts of an implant. Finally, we used a matched control to increase the power of the histopathological assessments. In summary, this method enables reliable and efficient discrimination and assessment of the retinal cytoarchitecture in an implanted eye.

Techniques for visualizing retinal cytoarchitecture directly adjacent to individual electrodes within a retinal stimulator.

With  the recent development of retinal prostheses, it is important to develop  reliable techniques for assessing the safety of these devices in ...

Synthesis of Soft Polysiloxane-urea Elastomers for Intraocular Lens Application

This study discusses a synthesis route for soft polysiloxane-based urea (PSU) elastomers for their applications as accommodating intraocular lenses (a-IOLs). Aminopropyl-terminated polydimethylsiloxanes (PDMS) were previously prepared via the ring-chain equilibration of the cyclic siloxane octamethylcyclotetrasiloxane (D4) and 1,3-bis(3-aminopropyl)-tetramethyldisiloxane (APTMDS). Phenyl groups were introduced into the siloxane backbone via the copolymerization of D4 and 2,4,6,8-tetramethyl-2,4,6,8-tetraphenyl-cyclotetrasiloxane (D4Me,Ph). These polydimethyl-methyl-phenyl-siloxane-block copolymers were synthesized for increasing the refractive indices of polysiloxanes. For applications as an a-IOL, the refractive index of the polysiloxanes must be equivalent to that of a young human eye lens. The polysiloxane molecular weight is controlled by the ratio of the cyclic siloxane to the endblocker APTMDS. The transparency of the PSU elastomers is examined by the transmittance measurement of films between 200 and 750 nm, using a UV-Vis spectrophotometer. Transmittance values at 750 nm (upper end of the visible spectrum) are plotted against the PDMS molecular weight, and &#62; 90% of the transmittance is observed until a molecular weight of 18,000 g&#183;mol&#8722;1. Mechanical properties of the PSU elastomers are investigated using stress-strain tests on die-cut dog-bone-shaped specimens. For evaluating mechanical stability, mechanical hysteresis is measured by repeatedly stretching (10x) the specimens to 5% and 100% elongation. Hysteresis considerably decreases with the increase in the PDMS molecular weight. In vitro cytotoxicity of some selected PSU elastomers is evaluated using an MTS cell viability assay. The methods described herein permit the synthesis of a soft, transparent, and noncytotoxic PSU elastomer with a refractive index approximately equal to that of a young human eye lens.

This study describes synthetic routes for aminopropyl-terminated polydimethylsiloxanes and polydimethyl-methyl-phenyl-siloxane-block copolymers and for soft polysiloxane-based urea (PSU) elastomers. It presents the application of PSUs as accommodating an intraocular lens. An evaluation method for in vitro cytotoxicity is also described.

This study discusses a synthesis route for soft polysiloxane-based urea (PSU) elastomers for their applications as accommodating intraocular lenses ...

Chemistry

Fabrication of Refractive-index-matched Devices for Biomedical Microfluidics

The use of microfluidic devices has emerged as a defining tool for biomedical applications. When combined with modern microscopy techniques, these devices can be implemented as part of a robust platform capable of making simultaneous complementary measurements. The primary challenge created by the combination of these two techniques is the mismatch in refractive index between the materials traditionally used to make microfluidic devices and the aqueous solutions typically used in biomedicine. This mismatch can create optical artifacts near the channel or device edges. One solution is to reduce the refractive index of the material used to fabricate the device by using a fluorinated polymer such as MY133-V2000 whose refractive index is similar to that of water (n = 1.33). Here, the construction of a microfluidic device made out of MY133-V2000 using soft lithography techniques is demonstrated, using O2 plasma in conjunction with an acrylic holder to increase the adhesion between the MY133-V2000 fabricated device and the polydimethylsiloxane (PDMS) substrate. The device is then tested by incubating it filled with cell culture media for 24 h to demonstrate the ability of the device to maintain cell culture conditions during the course of a typical imaging experiment. Finally, quantitative phase microscopy (QPM) is used to measure the distribution of mass within the live adherent cells in the microchannel. This way, the increased precision, enabled by fabricating the device from a low index of refraction polymer such as MY133-V2000 in lieu of traditional soft lithography materials such as PDMS, is demonstrated. Overall, this approach for fabricating microfluidic devices can be readily integrated into existing soft lithography workflows in order to reduce optical artifacts and increase measurement precision.

This protocol describes the fabrication of microfluidic devices from MY133-V2000 to eliminate artifacts that often arise in microchannels due to the mismatching refractive indices between microchannel structures and an aqueous solution. This protocol uses an acrylic holder to compress the encapsulated device, improving adhesion both chemically and mechanically.

The use of microfluidic devices has emerged as a defining tool for biomedical applications. When combined with modern microscopy techniques, these devices ...

Engineering

Development of an In Vitro Ocular Platform to Test Contact Lenses

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic physiological tear volumes.7 The traditional model also lacks the natural tear flow component and the blinking reflex, both of which are defining factors of the ocular environment. The development of a novel model is described in this study, which consists of a unique 2-piece design, eyeball and eyelid piece, capable of mimicking physiological tear volume. The models are created from 3-D printed molds (Polytetrafluoroethylene or Teflon molds), which can be used to generate eye models from various polymers, such as polydimethylsiloxane (PDMS) and agar. Further modifications to the eye pieces, such as the integration of an explanted human or animal cornea or human corneal construct, will permit for more complex in vitro ocular studies. A commercial microfluidic syringe pump is integrated with the platform to emulate physiological tear secretion. Air exposure and mechanical wear are achieved using two mechanical actuators, of which one moves the eyelid piece laterally, and the other moves the eyeballeyepiece circularly. The model has been used to evaluate CLs for drug delivery and deposition of tear components on CLs.

Development of an In Vitro Ocular Platform to Test Contact Lenses

Current in vitro models for evaluating contact lenses (CLs) and other eye-related applications are severely limited. The presented ocular platform simulates physiological tear flow, tear volume, air exposure and mechanical wear. This system is highly versatile and can be applied to various in vitro analyses with CLs.

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic ...

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical methods for manipulating and monitoring the activity of neurons in vivo. These types of experiments rely on two main components: 1) implantable devices that provide optical access to the brain, and 2) light-sensitive proteins that change neuronal excitability or provide a readout of neuronal activity. There are a number of ways to express light-sensitive proteins, but stereotaxic injection of viral vectors is currently the most flexible approach because expression can be controlled with genetic, anatomical, and temporal precision. Despite the great utility of viral vectors, delivering the virus to the site of optical implants poses numerous challenges. Stereotaxic virus injections are demanding surgeries that increase surgical time, increase the cost of studies, and pose a risk to the animal's health. The surrounding tissue can be physically damaged by the injection syringe, and by immunogenic inflammation caused by the abrupt delivery of a bolus of high-titer virus. Aligning injections with optical implants is especially difficult when targeting small regions deep in the brain. To overcome these challenges, we describe a method for coating multiple types of optical implants with films composed of silk fibroin and Adeno-associated viral (AAV) vectors. Fibroin, a polymer derived from the cocoon of Bombyx mori, can encapsulate and protect biomolecules and can be processed into forms ranging from soluble films to ceramics. When implanted into the brain, silk/AAV coatings release virus at the interface between optical elements and the surrounding brain, driving expression precisely where it is needed. This method is easily implemented and promises to greatly facilitate in vivo studies of neural circuit function.

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

Here, we present a method for delivering viral expression vectors into the brain using silk fibroin films. This method allows targeted delivery of expression vectors using silk/AAV coated optical fibers, tapered optical fibers, and cranial windows.

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical ...

Neuroscience

Microfabrication of Implantable Optics Integrated in a Microstructured Imaging Window for Advanced In Vivo Imaging

In the context of biomaterials and drug testing in animal models, this study presents a streamlined protocol for fabricating a novel implantable integrated imaging window. The micro-device comprises a sophisticated system of microlenses coupled with micro-scaffolds specifically designed for in vivo quantification of the immune response using advanced non-linear excitation microscopy. The protocol is based on two-photon polymerization (2PP) of the biocompatible photoresist SZ2080, which enables the fabrication of micro-scaffolds and micro-lenses in a continuous sequence to enhance manufacturing efficiency and precision. To further improve speed, accuracy, and structural integrity, a hybrid optics fabrication approach was implemented, involving the 2PP of the microlens outer shell followed by UV bulk crosslinking of the inner core. This innovative technique optimizes the optical properties of the microlenses while streamlining the production process. The resulting micro-device demonstrates high reproducibility and mechanical stability, making it an effective method for prototyping microscale optical systems for a range of biomedical applications.

This protocol describes the fabrication of an implantable integrated imaging window using 3D laser printing. The window consists of a system of microlenses coupled with micro-scaffolds. The method involves two-photon polymerization (2PP) of the biocompatible photoresist SZ2080 in a continuous sequence, optimizing manufacturing efficiency and alignment between the different components.

In the context of biomaterials and drug testing in animal models, this study presents a streamlined protocol for fabricating a novel implantable ...

Evaluating Optokinetic Reflex Responses in Mice: A New High-Precision Protocol

Quantification of Visual Feature Selectivity of the Optokinetic Reflex in Mice

The optokinetic reflex (OKR) is an essential innate eye movement that is triggered by the global motion of the visual environment and serves to stabilize retinal images. Due to its importance and robustness, the OKR has been used to study visual-motor learning and to evaluate the visual functions of mice with different genetic backgrounds, ages, and drug treatments. Here, we introduce a procedure for evaluating OKR responses of head-fixed mice with high accuracy. Head fixation can rule out the contribution of vestibular stimulation on eye movements, making it possible to measure eye movements triggered only by visual motion. The OKR is elicited by a virtual drum system, in which a vertical grating presented on three computer monitors drifts horizontally in an oscillatory manner or unidirectionally at a constant velocity. With this virtual reality system, we can systematically change visual parameters like spatial frequency, temporal/oscillation frequency, contrast, luminance, and the direction of gratings, and quantify tuning curves of visual feature selectivity. High-speed infrared video-oculography ensures accurate measurement of the trajectory of eye movements. The eyes of individual mice are calibrated to provide opportunities to compare the OKRs between animals of different ages, genders, and genetic backgrounds. The quantitative power of this technique allows it to detect changes in the OKR when this behavior plastically adapts due to aging, sensory experience, or motor learning; thus, it makes this technique a valuable addition to the repertoire of tools used to investigate the plasticity of ocular behaviors.

Author Spotlight: An Accurate and Quantitative Approach to Study Visual Feature Selectivity of the Optokinetic Reflex in Mice 

Here, we describe a standard protocol for quantifying the optokinetic reflex. It combines virtual drum stimulation and video-oculography, and thus allows precise evaluation of the feature selectivity of the behavior and its adaptive plasticity.

The optokinetic reflex (OKR) is an essential innate eye movement that is triggered by the global motion of the visual environment and serves to stabilize ...

Cryosectioning and Immunostaining of Mouse Retina

Tissue sectioning and immunohistochemistry are essential techniques in histological and pathological studies of retinal diseases using animal models. These methods enable detailed examinations of tissue morphologies and the localization of specific proteins within the tissue, which provide valuable insights into disease processes and mechanisms. Mice are the most widely used model for this purpose. However, because mouse eyeballs are small and mouse retinas are extremely delicate tissues, obtaining high-quality retinal sections and immunostaining images from mouse eyeballs is typically challenging. This study describes an improved protocol for cryosectioning mouse retinas and performing immunohistochemistry. An essential point of this protocol involves coating the eyeball with a layer of super glue, which prevents deformation of the eyeballs during the processes of cornea removal, lens extraction, and embedding. This step ensures the integrity of retinal morphologies is well preserved. This protocol highlights critical technical considerations and optimization strategies for consistently producing high-quality retinal sections and achieving excellent immunostaining results.

A protocol for preparing mouse retinal cryosections and performing immunostaining on photoreceptors is described. This article enables researchers to consistently produce mouse retinal frozen sections with well-preserved morphology and high-quality immunostaining results.

Tissue sectioning and immunohistochemistry are essential techniques in histological and pathological studies of retinal diseases using animal models. ...

Rabbit Eye Dissection and Cryosectioning for Immunohistochemistry Studies

Obtaining High-Quality Cryosections of Whole Rabbit Eye

This protocol describes how to obtain high-quality retinal cryosections in larger animals, such as rabbits. After enucleation, the eye is briefly immersed in the fixative. Then, the cornea and iris are removed and the eye is left overnight for additional fixation at 4 &#176;C. Following fixation, the lens is removed. The eye is then placed in a cryomold and filled with an embedding medium. By removing the lens, the embedding medium has better access to the vitreous and leads to better retinal stability. Importantly, the eye should be incubated in embedding medium overnight to allow complete infiltration throughout the vitreous. Following overnight incubation, the eye is frozen on dry ice and sectioned. Whole retinal sections may be obtained for use in immunohistochemistry. Standard staining protocols may be utilized to study the localization of antigens within the retinal tissue. Adherence to this protocol results in high-quality retinal cryosections that may be used in any experiment utilizing immunohistochemistry.

Author Spotlight: Optimizing Retinal Tissue Processing for Immunohistochemistry in Rabbit Models

This protocol describes a reliable method for obtaining high-quality cryosections of whole rabbit eyes. It details rabbit eye dissection, fixation, embedding, and sectioning procedures, which may be easily adapted for use in any study utilizing immunohistochemistry in larger eyes.

This protocol describes how to obtain high-quality retinal cryosections in larger animals, such as rabbits. After enucleation, the eye is briefly immersed ...

Customizing a Cryolite Glass Prosthetic Eye

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