Os seguintes protocolos são aceitos sob cuidado de Animal institucional da Universidade de Maryland e Comissão de utilização, bem como o Conselho de revisão institucional. Os protocolos seguem normas federais, estaduais e locais, e as diretrizes estabelecem pela política de Universidade de Maryland em biossegurança.
1. dissecação da amostra de olho
<ol><li>
Obter uma amostra de olho do banco local de matadouro ou tecido. Se um globo de olho inteiro é obtido, extrair-se imediatamente a lente, zónulas anexadas e vítreo. Nota: Os detalhes específicos descritos a seguir referem-se aos olhos dos suínos e humanos.<ol>
<li>Usando a tesoura cirúrgica desinfectada e fórceps, cortar e remover todo o tecido em excesso em torno da esclera.</li>
		<li>Firmemente manter o olho em seu lado e, usando uma lâmina de barbear, faça uma pequena incisão ao longo do lado do olho 3 mm longe da córnea. Fazer a corte profunda o suficiente para ter atingido o vítreo dentro do olho.</li>
		<li>Com uma tesoura, corte com cuidado mais longo a incisão ao redor da circunferência do olho. Evite perfurar a lente. Uma imagem representativa é mostrada na Figura 2.</li>
		<li>Uma vez que a circunferência externa do olho foi cortada, remova o tecido posterior do olho usando fórceps. Isole a lente, zónulas, corpo ciliar e o vítreo anexado com fórceps. Uma imagem representativa é mostrada na Figura 2B.</li>
		<li>Usando a tesoura e pinça, remova o vítreo em excesso para que a lente pode deitado na MLS. Nota: Em casos de transplante de córnea, o botão corneano é usado na cirurgia e o restante do globo está disponível para fins de investigação. No entanto, este globo parcial ainda pode ser usado na preparação da instalação do esticador de lente tecido. Se apenas a parte posterior é obtida, apenas execute o passo 1.1.4–1.1.5.</li>
	</ol>
</li>
	<li>Desinfecte todos pós-dissecção de equipamentos usados em solução de água sanitária de 15% por 30 min.</li>
</ol>2. julgamento montagem da lente Manual maca
<ol><li>Insira as solas de sapato de 10 mm e os topos de sapato correspondente na placa inferior da MLS então ainda há uma lacuna de 5 mm entre a parede traseira do travessão, sapato e o sapato em si.</li>
	<li>Alinhar as placas superior e inferior, encaixe as placas juntos; o dispositivo está agora na posição unstretched.</li>
	<li>Insira as placas no caso a placa e o parafuso de bloqueio no orifício localizado na parte lateral da placa de fundo.</li>
	<li>Insira o caso da placa na base e coloque a chave para os travessões alinhados.</li>
	<li>Gire a chave no sentido horário até atingir o parafuso de parada para os sapatos do contrato e torcer volta anti-horário para devolver os sapatos para a posição original unstretched.</li>
</ol>3. montagem da lente
<ol><li>Insira as solas de sapato de 10 mm na placa inferior na MLS para que um 5mm gap entre restos a parede traseira do travessão o sapato e o sapato em si.</li>
	<li>Usando pinças curvas, coloque o lente extraído virado para cima no meio da placa de fundo para que os sapatos estão apoiando a lente sobre o furo central.</li>
	<li>Encaixe o topo correspondente dos sapatos no lugar, recorte apenas as zónulas e o vítreo. Visualmente, assegurar que a lente se mantém como centrado possível na placa inferior.</li>
	<li>Repita as etapas 2.3 – 2.4.</li>
</ol>4. medição da lente
<ol><li>Coloca um sistema de imagem diretamente acima do aparelho para capturar vídeos e imagens do processo de alongamento. Não se esqueça de incluir uma régua na moldura do quadro com precisão imagens de tamanho e escala no pós-processamento. Nota: Qualquer sistema de imagem adequado é suficiente para esta etapa; Aqui usamos um 12 megapixel, focagem automática smartphone um pé da amostra.</li>
	<li>Com firmeza, mas suavemente, gire a chave no sentido horário para esticar a lente. A Figura 3 mostra imagens representativas no estado unstretched e esticada.</li>
	<li>Depois de fotografar a lente esticada, gire a chave no sentido anti-horário para restaurar a amostra em seu estado de repouso. Nota: É imperativo que a medida da lente é realizada em tempo hábil para minimizar a desidratação da lente.</li>
	<li>Fotografe claramente o estado de repouso final da lente.</li>
</ol>5. análise de dados
<ol><li>Fazer o upload da imagem para ImageJ e, use o recurso de "ponto" para selecionar pelo menos 40 pontos em torno da circunferência da lente conforme mostrado na Figura 4. Use a opção de "Medir" → "Analisar" para produzir a localização de cada ponto selecionado.</li>
	<li>Pontos de ajuste (usando o software , por exemplo, MATLAB) os local a fim de produzir um raio e qui-quadrado do ajuste conforme mostrado na Figura 4B. Converta o pixel radius e erro em métricas usando a régua fotografada.</li>
	<li>Execute duas caudas teste t pareado para comparar uma lente individual antes e depois alongamento da MLS.</li>
</ol>

<ol>
	<li>Von Helmholtz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uber+die+akkommodation+des+auges.">Uber die akkommodation des auges.</a> Arch Ophthal. 1, 1-74 (1855).</li><li>Schachar, R. A., Black, T. D., Kash, R. L., Cudmore, D. P., Schanzlin, D. 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+mechanism+of+accommodation+and+presbyopia+in+the+primate.">The mechanism of accommodation and presbyopia in the primate.</a> Ann Ophthalmol. 27, 58-67 (1995).</li><li>Glasser, A., Campbell, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presbyopia+and+the+optical+changes+in+the+human+crystalline+lens+with+age.">Presbyopia and the optical changes in the human crystalline lens with age.</a> Vision Res. 38 (2), 209-229 (1998).</li><li>Reilly, M. A., Hamilton, P. D., Perry, G., Ravi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+behavior+and+natural+and+refilled+porcine+lenses+in+a+robotic+lens+stretcher.">Comparison of the behavior and natural and refilled porcine lenses in a robotic lens stretcher.</a> Exp Eye Res. 88, 483-494 (2009).</li><li>Langenbucher, A., Huber, S., Nguyen, N. X., Seitz, B., Gusek-Schneider, G. C., K&#252;chle, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+accommodation+after+implantation+of+an+accommodating+posterior+chamber+intraocular+lens.">Measurement of accommodation after implantation of an accommodating posterior chamber intraocular lens.</a> J Cataract Refract Surg. 29 (4), 677-685 (2003).</li><li>Ehrmann, K., Ho, A., Parel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+analysis+of+the+accommodative+apparatus+in+primates.">Biomechanical analysis of the accommodative apparatus in primates.</a> Clin Exp Optom. 91 (4), 411 (2008).</li><li>Pinilla Cort&#233;s, L., 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=Experimental+Protocols+for+Ex+Vivo+Lens+Stretching+Tests+to+Investigate+the+Biomechanics+of+the+Human+Accommodation+Apparatus.">Experimental Protocols for Ex Vivo Lens Stretching Tests to Investigate the Biomechanics of the Human Accommodation Apparatus.</a> Invest Ophthalmol Vis Sci. 56 (5), 2926 (2015).</li><li>Fisher, R. 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+elastic+constants+of+the+human+lens.">The elastic constants of the human lens.</a> J Physiol. 212 (1), 147-180 (1971).</li><li>Eppig, 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=Biomechanical+eye+model+and+measurement+setup+for+investigating+accommodating+intraocular+lenses.">Biomechanical eye model and measurement setup for investigating accommodating intraocular lenses.</a> Z Med Ohys. 23 (2), 144-152 (2013).</li><li>Manns, F., Parel, , 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=Response+of+Human+and+Monkey+Lenses+in+a+Lens+Stretcher.">Response of Human and Monkey Lenses in a Lens Stretcher.</a> Invest Ophthalmol Vis Sci. 48 (7), 3260 (2007).</li><li>Scarcelli, G., Kim, P., Yun, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+age-related+stiffening+in+the+crystalline+lens+by+Brillouin+optical+microscopy.">In vivo measurement of age-related stiffening in the crystalline lens by Brillouin optical microscopy.</a> Biophys J. 101 (6), 1539-1545 (2011).</li><li>Besner, S., Scarcelli, G., Pineda, R., Yun, S. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Brillouin+Analysis+of+the+Aging+Crystalline+Lens.">In Vivo Brillouin Analysis of the Aging Crystalline Lens.</a> Invest Ophthalmol Vis Sci. 57 (13), 5093 (2016).</li><li>Cortes, L., 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=Experimental+Protocols+for+Ex+Vivo+Lens+Stretching+Tests+to+Investigate+the+Biomechanics+of+the+Human+Accommodation+Apparatus.">Experimental Protocols for Ex Vivo Lens Stretching Tests to Investigate the Biomechanics of the Human Accommodation Apparatus.</a> Invest Ophthalmol Vis Sci. 56 (5), 2926-2932 (2015).</li><li>Bernal, A., Parel, J. -. M., Manns, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+Posterior+Zonular+Fiber+Attachment+on+the+Anterior+Hyaloid+Membrane.">Evidence for Posterior Zonular Fiber Attachment on the Anterior Hyaloid Membrane.</a> Invest Ophthalmol Vis Sci. 47 (11), 4708 (2006).</li><li>Kammel, R., Ackermann, R., Mai, T., Damm, C., Nolte, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pig+Lenses+in+a+Lens+Stretcher.">Pig Lenses in a Lens Stretcher.</a> Optom Vis Sci. 89 (6), 908-915 (2012).</li><li>Hahn, 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=Measurement+of+Ex+Vivo+Porcine+Lens+Shape+During+Simulated+Accommodation,+Before+and+After+fs-Laser+Treatment.">Measurement of Ex Vivo Porcine Lens Shape During Simulated Accommodation, Before and After fs-Laser Treatment.</a> Invest Ophthalmol Vis Sci. 56 (9), 5332-5343 (2015).</li><li>D'Antin, J. C., Cortes, L. P., Montenegro, G. A., Barraquer, R. I., Michael, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+portable+manual+stretching+device+to+simulate+accommodation.">Evaluation of a portable manual stretching device to simulate accommodation.</a> Acta Ophthalmol. 93 (255), (2015).</li><li>Pierscionek, B. <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+response+of+human+lenses+to+stretching+forces.">Age-related response of human lenses to stretching forces.</a> Exp Eye Res. 60 (3), 325-332 (1995).</li><li>Marussich, L., 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=Measurement+of+Crystalline+Lens+Volume+During+Accommodation+in+a+Lens+Stretcher.">Measurement of Crystalline Lens Volume During Accommodation in a Lens Stretcher.</a> Invest Ophthalmol Vis Sci. 56 (8), 4239 (2015).</li><li>Martinez-Enriquez, E., P&#233;rez-Merino, P., Velasco-Ocana, M., Marcos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OCT-based+full+crystalline+lens+shape+change+during+accommodation+in+vivo.">OCT-based full crystalline lens shape change during accommodation in vivo.</a> Biomed Opt Exp. 8 (2), 918-933 (2017).</li></ol>

AB tem participação no Bioniko Consulting LLC.

Nós desenvolvemos um novo método para fornecer uma maneira precisa e eficiente de estudar a capacidade de alojamento da lente, utilizando um mecanismo de fixação de duplo-peça para acoplar a maca para a amostra. Durante o alojamento, a lente relaxa, e o diâmetro diminui em resposta ao relaxamento de fibras zonular1,2,4,19. O método centra-se sobre este fenómeno de aperto e controlando a...

A acomodação é o processo pelo qual o olho humano é capaz de ajustar dinamicamente a forma de seu cristalino para ver objetos a distâncias longe ou perto, em foco. Acomodação é um processo intrinsecamente biomecânico. Após estímulo neural, os músculos ciliares produzem uma força sobre o corpo ciliar e das fibras de zonule que atribuem a circunferência da lente da cápsula1,2. Embora existam diferentes teorias atrás a biomecânica de alojamento, a mais amplamente aceita é a hipótese de Helmholtz. De acordo com a hipótese, a lente está em um estado natural e esticado, correspondente à forma mais fina da lente que é ideal para o foco de objetos distantes. Para alterar o foco para objetos mais próximos, contraem os músculos ciliares e as fibras zonular estão relaxadas. Por sua vez, engrossa a lente, aumentando a curvatura da superfície anterior e posterior. Isso corresponde a um aumento no poder dióptrico que é necessário para a visão, portanto, uma menor distância focal1de perto.
A capacidade de acomodar é comprometida ao longo do tempo através de uma condição chamada presbiopia. Afetando todo mundo por 50 anos, presbiopia faz com que o olho não é possível alterar dinamicamente o foco de longe para fechar distâncias3. Para combater a presbiopia, métodos atuais são passivos incluindo lentes bifocais e lentes corretivas. Ao mesmo tempo aumentar a habilidade de focalizar objetos próximos em alguns aviões, tais tratamentos passivos não é possível restaurar a capacidade dinâmica de foco da lente,4,5. A fim de tratar a presbiopia eficientemente, ou possivelmente impedi-lo, há uma necessidade contínua para entender melhor o alojamento.
Para estudar a lente alojamento, um número de dispositivos foram desenvolvido para simular o fenômeno ex vivo4,6,7,8,9. Foram introduzidos, para monitorar o alongamento da lente através de de forças centrífugas8discos girando. Para replicar mais fielmente o fenômeno, lente alongamento dispositivos foram gradualmente introduzidos e inovou. Usando uma lente maca, Manns et al. caracterizada a força necessária para acomodar a lente enquanto correlacionando tal a potência da lente e o diâmetro equatorial9. Entendimento atual é que a lente endurece com a idade, resultando em uma mudança reduzida em forma de lente, em resposta a uma força igual a partir do corpo ciliar3,10,11,12.
Macas de lente atual muitas vezes envolvem uma configuração complexa, implementando eletrônica e taxas de alongamentos programáveis e requer o cadáver inteiro globo ocular6,7,10,13. Esta exigência aumenta o custo por experiência de mais de US $500,00 por olho e disponibilidade de amostra. Aqui nós apresentamos um método para replicar a lente alojamento a baixo custo, como o olho posterior totaliza cerca US $200,00. Pouco menos sofisticado do que muitos dispositivos usados hoje, a técnica é muito mais custo-eficaz e adotáveis sem comprometer os resultados. Este método é centrado em torno de uma maca de lente manual (MLS), representada na Figura 1e usa um sistema exclusivo de fixação sobre as fibras zonular e um método de torção radial para expandir o diâmetro da lente. A precisão fisiológica do protocolo é validada pelas conclusões do Bernal et al., que estudou o caminho pelo qual as fibras zonular anteriores e posteriores estão ligadas para a cápsula de lente14. Usando o design de sapatos personalizados, que exigem apenas a lente, zonule e corpo ciliar, objetivamos estudar biomecânica lente replicando Alojamento fisiológico.

<table><tbody><tr><td>Manual Lens Stretcher</td><td>Bioniko</td><td>MLS</td><td>Different animal species will require different shoe sizes</td></tr><tr><td>Porcine Eye Samples</td><td>George G. Ruppersberger; slaughterhouse</td><td>N/A</td><td>Whole eyeballs were obtained</td></tr><tr><td>Human Eye Samples</td><td>The National Disease Research Interchange</td><td>N/A</td><td>Posterior poles without corneas were ordered</td></tr><tr><td>Dissecting Scissors (5 1/2&#039;&#039; Straight)</td><td>Electron Microsopy Sciences</td><td>72960</td><td></td></tr><tr><td>Tissue Forceps (4 1/2&#039;&#039;)</td><td>Electron Microsopy Sciences</td><td>72960</td><td></td></tr><tr><td>iPhone 6s</td><td>Apple</td><td>N/A</td><td>Any imaging system with ~0.1 mm resolution will work</td></tr><tr><td>Sodium Hypochorite</td><td>Clorox</td><td>Clorox Regular-Bleach</td><td>Any disinfectant will work</td></tr></tbody></table>

simulating the mechanics of lens accommodation via a manual lens stretcher

O objetivo do presente protocolo é imitar a biomecânica de acomodação fisiológica de uma maneira custo-eficiente e prática. Alojamento é alcançado através da contração do corpo ciliar e relaxamento das fibras zonule, que resulta em espessamento da lente necessário para visão de perto. Aqui, apresentamos um romance, um método simples, no qual alojamento é replicado por enrijecer as zónulas ligadas da cápsula da lente através de um esticador de lente manual (MLS). Esse método monitora o alongamento radial alcançado por uma lente quando submetido a uma força consistente e permite uma comparação de acomodar lentes, que podem ser esticadas, a não-acomodando lentes, que não podem ser esticadas. Importante, os casais de maca para as zónulas diretamente e não para a esclera do olho, exigindo apenas a lente, zónulas e corpo ciliar ao invés da amostra de todo o globo. Esta diferença pode diminuir significativamente o custo de aquisição de lentes de dador cadáver cerca de 62% em relação a aquisição de todo o globo.

Olhos de suínos, uma amostra comum para estudar a presbiopia através de lente alongamento4,15, obtiveram-se, (n = 10) de um matadouro local e este protocolo foi utilizado para observar a capacidade de alojamento das lentes. Figura 5 A mostra a comparação da lente suínos antes e após alongamento através da MLS. Houve um aumento de 0,07 mm média 0,19 ± raio de lente quando esti...

Watch this Scientific Journal Video about Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher at JoVE.com

Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher

Simulando a mecânica de lente Alojamento através de uma lente Manual esticador

Apresentamos um método eficiente de estudar Alojamento lente usando uma maca de lente manual. A imita protocolo fisiológico Alojamento puxando as zónulas ligado ao redor da cápsula da lente, desse modo, estendendo-se a lente.

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved ...

simulating-mechanics-lens-accommodation-via-manual-lens

Research

JoVE Journal

Bioengineering

6.7K visualizações.  University of Maryland. Apresentamos um método eficiente de estudar Alojamento lente usando uma maca de lente manual. A imita protocolo fisiológico Alojamento puxando as zónulas ligado ao redor da cápsula da lente, desse modo, estendendo-se a lente.

Vídeo: Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher

Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers

Single-molecule techniques for stretching DNA of contour lengths less than a kilobase are fraught with experimental difficulties. However, many interesting biological events such as histone binding and protein-mediated looping of DNA1,2, occur on this length scale. In recent years, the mechanical properties of DNA have been shown to play a significant role in fundamental cellular processes like the packaging of DNA into compact nucleosomes and chromatin fibers3,4. Clearly, it is then important to understand the mechanical properties of short stretches of DNA. In this paper, we provide a practical guide to a single-molecule optical tweezing technique that we have developed to study the mechanical behavior of DNA with contour lengths as short as a few hundred basepairs.
The major hurdle in stretching short segments of DNA is that conventional optical tweezers are generally designed to apply force in a direction lateral to the stage5,6, (see Fig. 1). In this geometry, the angle between the bead and the coverslip, to which the DNA is tethered, becomes very steep for submicron length DNA. The axial position must now be accounted for, which can be a challenge, and, since the extension drags the microsphere closer to the coverslip, steric effects are enhanced. Furthermore, as a result of the asymmetry of the microspheres, lateral extensions will generate varying levels of torque due to rotation of the microsphere within the optical trap since the direction of the reactive force changes during the extension.
Alternate methods for stretching submicron DNA run up against their own unique hurdles. For instance, a dual-beam optical trap is limited to stretching DNA of around a wavelength, at which point interference effects between the two traps and from light scattering between the microspheres begin to pose a significant problem. Replacing one of the traps with a micropipette would most likely suffer from similar challenges. While one could directly use the axial potential to stretch the DNA, an active feedback scheme would be needed to apply a constant force and the bandwidth of this will be quite limited, especially at low forces.
We circumvent these fundamental problems by directly pulling the DNA away from the coverslip by using a constant force axial optical tweezers7,8. This is achieved by trapping the bead in a linear region of the optical potential, where the optical force is constant-the strength of which can be tuned by adjusting the laser power. Trapping within the linear region also serves as an all optical force-clamp on the DNA that extends for nearly 350 nm in the axial direction. We simultaneously compensate for thermal and mechanical drift by finely adjusting the position of the stage so that a reference microsphere stuck to the coverslip remains at the same position and focus, allowing for a virtually limitless observation period.

We illustrate the use of a constant force axial optical tweezers to explore the mechanical properties of short DNA molecules. By stretching DNA axially, we minimize steric hindrances and artifacts arising in conventional lateral manipulation, allowing us to study DNA molecules as short as ~100 nm.

Alongamento curtas seqüências de DNA com Pinça força constante óptico axial

Single-molecule techniques for stretching DNA of contour lengths  less than a kilobase are fraught with experimental difficulties. However, many ...

Bioengenharia

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

Alongamento Células micropatterned num PDMS Membrana

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

Uma plataforma Novel Alongamento para Aplicações em Celular e Tecidual mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

Live Cell Imaging during Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

Imagens de células vivas durante Mecânica estiramento

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited 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.

Síntese de elastômeros de silicone macio-ureia para aplicação de lente intra-ocular

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

Química

Production of a Strain-Measuring Device with an Improved 3D Printer

A traditional strain measurement sensor needs to be electrified and is susceptible to electromagnetic interference. In order to solve the fluctuations in the analog electrical signal in a traditional strain gauge operation, a new strain measurement method is presented here. It uses a photographic technique to display the strain change by amplifying the change of the pointer displacement of the mechanism. A visual polydimethylsiloxane (PDMS) lens with a focal length of 7.16 mm was added to a smartphone camera to generate a lens group acting as a microscope to capture images. It had an equivalent focal length of 5.74 mm. Acrylonitrile butadiene styrene (ABS) and nylon amplifiers were used to test the influence of different materials on the sensor performance. The production of the amplifiers and PDMS lens is based on improved 3D printing technology. The data obtained were compared with the results from finite element analysis (FEA) to verify their validity. The sensitivity of the ABS amplifier was 36.03 &plusmn; 1.34 &#181;&epsilon;/&#181;m, and the sensitivity of the nylon amplifier was 36.55 &plusmn; 0.53 &#181;&epsilon;/&#181;m.

This work presents a strain measurement sensor consisting of an amplification mechanism and a polydimethylsiloxane microscope manufactured using an improved 3D printer.

Produção de um dispositivo de medição de tensão com uma impressora 3D melhorada

A traditional strain measurement sensor needs to be electrified and is susceptible to electromagnetic interference. In order to solve the fluctuations in ...

Engenharia

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

Tensão controlada de hidrogéis 3D sob imagem de microscopia ao vivo

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Caracterização Biomecânica de Lente Ocular Suína com Compressão Automatizada

Automated Compression Testing of the Ocular Lens

The biomechanical properties of the ocular lens are essential to its function as a variable power optical element. These properties change dramatically with age in the human lens, resulting in a loss of near vision called presbyopia. However, the mechanisms of these changes remain unknown. Lens compression offers a relatively simple method for assessing the lens' biomechanical stiffness in a qualitative sense and, when coupled with appropriate analytical techniques, can help quantify biomechanical properties. A variety of lens compression tests have been performed to date, including both manual and automated, but these methods inconsistently apply key aspects of biomechanical testing such as preconditioning, loading rates, and time between measurements. This paper describes a fully automated lens compression test wherein a motorized stage is synchronized with a camera to capture the force, displacement, and shape of the lens throughout a preprogrammed loading protocol. A characteristic elastic modulus may then be calculated from these data. While demonstrated here using porcine lenses, the approach is appropriate for the compression of lenses of any species.

Autor em destaque: Avançando na compreensão das mudanças de rigidez da lente relacionadas à idade 

We present an automated method for characterizing the effective elastic modulus of an ocular lens using a compression test.

Teste automatizado de compressão da lente ocular

The biomechanical properties of the ocular lens are essential to its function as a variable power optical element. These properties change dramatically ...

Simulando a mecânica de lente Alojamento através de uma lente Manual esticador

Resumo

Explore mais vídeos

PLAYLIST