Se aceptan los siguientes protocolos bajo cuidado de Animal institucional de la Universidad de Maryland y Comisión de uso así como la Junta de revisión institucional. Los protocolos siguen las normas federales, estatales y locales, y las directrices establecidas por la política de la Universidad de Maryland sobre bioseguridad.
1. disección de ojo muestra
<ol><li>
Obtener una muestra ojo banco tejido o matadero local. Si se obtiene un globo de todo ojo, extraiga inmediatamente la lente zonules adjuntos y vítreo. Nota: Los detalles específicos que se describen a continuación pertenecen a ojos porcinos y humanos.<ol>
<li>Utilizando pinzas y tijeras quirúrgicas desinfectadas, corte y retire todo exceso de tejido que rodea la esclera.</li>
		<li>Con firmeza mantener el ojo en su lado y, usando una hoja de afeitar, hacer una pequeña incisión en el lado del ojo 3 mm de la córnea. Hacer el corte profundo suficiente haber alcanzado el vítreo dentro del ojo.</li>
		<li>Con unas tijeras, corte con cuidado más a lo largo de la incisión alrededor de la circunferencia del ojo. Evite perforar el objetivo. En la figura 2Ase muestra una imagen representativa.</li>
		<li>Una vez que se ha cortado la circunferencia exterior del ojo, eliminar el tejido posterior del ojo con unas pinzas. Aislar la lente, zonules, cuerpo ciliar y el vítreo atado con pinzas. Una imagen representativa se muestra en la figura 2B.</li>
		<li>Usando las tijeras y pinzas, quite exceso vítreo por lo que la lente puede poner plana en la MLS. Nota: En casos de trasplante de córnea, el botón corneal se utiliza en cirugía y el resto del mundo está disponible para fines de investigación. Sin embargo, este mundo parcial puedan utilizarse en la preparación del tejido de la configuración de la camilla de la lente. Si sólo se obtiene la parte posterior, sólo realizar paso 1.1.4–1.1.5.</li>
	</ol>
</li>
	<li>Desinfectar toda post-disección equipo usado en solución de lejía de 15% durante 30 minutos.</li>
</ol>2. montaje de prueba de la camilla de lente Manual
<ol><li>Inserte los fondos del zapato de 10 mm y las correspondientes tapas de zapato la placa inferior de la MLS por lo que queda un espacio de 5 mm entre la pared posterior del guión de zapato y la zapatilla en sí mismo.</li>
	<li>Alinee las placas superior e inferior, ajuste de las placas el dispositivo está ahora en la posición sin estiramiento.</li>
	<li>Inserte las placas en el caso de la placa y el tornillo de tope en el orificio situado en el lado de la placa inferior.</li>
	<li>Inserte la caja de la placa en la base y colocar la llave en los guiones alineados.</li>
	<li>Gire la llave en sentido horario hasta que llegue el tornillo de parada para contratar los zapatos y gire nuevamente hacia la izquierda para devolver los zapatos a la posición original sin estirar.</li>
</ol>3. montaje de la lente
<ol><li>Inserte el fondo del zapato de 10 mm en la placa inferior en la MLS para que una de 5 mm espacio entre restos la pared posterior de la sangría de zapato y zapato sí mismo.</li>
	<li>Con unas pinzas curvas, ponga la cara de la lente extraída hasta en el centro de la placa inferior para que los zapatos están apoyando la lente sobre el agujero central.</li>
	<li>Encajen el correspondiente superior de los zapatos, recortar sólo los zonules y el vítreo. Asegurarse visualmente que la lente permanece centrado posible en la placa inferior.</li>
	<li>Repita los pasos 2.3-2.4.</li>
</ol>4. medición de la lente
<ol><li>Coloque un sistema de proyección de imagen directamente encima del aparato para capturar vídeos y fotos del proceso de estiramiento. Asegúrese de incluir una regla en el marco de la imagen con precisión imágenes de tamaño y escala en post-proceso. Nota: Cualquier sistema de proyección de imagen adecuado es suficiente para este paso; Aquí utilizamos un 12 megapíxeles, enfoque automático smartphone un pie de la muestra.</li>
	<li>Con firmeza pero suavemente, gire la llave en sentido horario para estirar la lente. La figura 3 muestra imágenes representativas en el estado estirado y sin estirar.</li>
	<li>Después de fotografiar la lente se extendía, gire la llave en la dirección hacia la izquierda para restaurar la muestra en su estado de reposo. Nota: Es imprescindible que la medida de la lente se realiza de manera oportuna para reducir al mínimo la deshidratación de la lente.</li>
	<li>Claramente el estado final de reposo de la lente de la fotografía.</li>
</ol>5. Análisis de datos
<ol><li>Cargar la imagen en ImageJ y utilice la función de "punto" para seleccionar al menos 40 puntos alrededor de la circunferencia de la lente como se muestra en la figura 4A. Usa el "Analizar" → "Medida" para dar la ubicación de cada punto seleccionado.</li>
	<li>Ajuste (usando software por ejemplo, MATLAB) los puntos de ubicación con el fin de producir un radio y un Chi-cuadrado de ajuste como se muestra en la figura 4B. Convertir el radio de píxeles y el error en mediciones con la regla fotografiado.</li>
	<li>Realizar pares dos colas prueba t para comparar un lente individual antes y después de que se extiende desde la 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 tiene interés de propiedad en Bioniko Consulting LLC.

Hemos ideado un novedoso método para proporcionar una forma precisa y eficiente de estudiar la capacidad de alojamiento de la lente mediante la utilización de un mecanismo de sujeción de dos piezas para acoplar la camilla a la muestra. Alojamiento, la lente se relaja y disminuye el diámetro en respuesta a la relajación de las fibras zonulares1,2,4,19. El método se centra en este fenómeno...

El alojamiento es el proceso por el cual el ojo humano es capaz de ajustar dinámicamente la forma de su cristalino para ver objetos a distancias lejanas o cercanas en foco agudo. Es un proceso intrínsecamente biomecánico. Al estímulo de nervios, los músculos ciliares producen una fuerza sobre el cuerpo ciliar y las fibras de la zónula que se adhieren a la circunferencia de la lente cápsula1,2. Si bien existen diversas teorías detrás de la biomecánica del alojamiento, la más ampliamente aceptada es la hipótesis de Helmholtz. Según la hipótesis, la lente es en estado natural se extendía, correspondiente a la forma más fina de la lente que es óptima para el enfoque de objetos lejanos. Para cambiar el enfoque para objetos más cercanos, los músculos ciliares se contraen y se relajan las fibras zonulares. A su vez, la lente se espese, aumentando las curvaturas de la superficie anteriores y posteriores. Esto corresponde a un aumento de potencia dióptrica necesaria para cerca de visión, por lo tanto, un más corto longitud focal1.
La capacidad de acomodar se compromete con el tiempo a través de una condición llamada presbicia. Que afectan a todo el mundo por los 50 años, presbicia hace que el ojo no puede dinámicamente cambiar el enfoque de lejos a cerrar distancias3. Para combatir la presbicia, los métodos actuales son pasivos incluyendo bifocales y lentes correctivos. Aumentando la capacidad de enfocar objetos cercanos en pocos planos, tales tratamientos pasivos no pueden restaurar la capacidad de enfoque dinámico de la lente4,5. Para tratar la presbicia eficientemente, o posiblemente evitar que, hay una constante necesidad de entender mejor alojamiento.
Para estudiar el establecimiento de objetivo, ha desarrollado una serie de dispositivos para simular el fenómeno ex vivo4,6,7,8,9. Hace girar los discos primero fueron introducidos para controlar el estiramiento de la lente a través de fuerzas centrífugas8. Para reproducir más fielmente el fenómeno, dispositivos de estiramiento objetivo poco a poco se introdujo e innovados. Usando una lente camilla, Manns et al. caracteriza la fuerza requerida para acomodar la lente mientras la correlación de tal energía objetivo y diámetro ecuatorial9. Comprensión actual es que la lente endurece con la edad, lo que resulta en un cambio menor en forma de lente en respuesta a una fuerza igual de cuerpo ciliar3,10,11,12.
Camillas de lente actual a menudo implican una configuración compleja, aplicación electrónica y programables tipos de estiramientos y requiere el cadáver entero globo ocular6,7,10,13. Este requisito aumenta el coste del experimento a más de $500,00 por ojo y disminuye la disponibilidad de la muestra. Aquí presentamos un método para replicar lente alojamiento a bajo costo como el ojo posterior total alrededor de $200,00. Mientras menos sofisticado que muchos de los dispositivos utilizados en la actualidad, la técnica es mucho más rentable y adoptables sin comprometer resultados. Este método se centra en una camilla de lente manual (MLS) representada en la figura 1y utiliza un sistema único de sujeción en las fibras zonulares y un método de torsión radial para ampliar el diámetro de la lente. La precisión fisiológica del protocolo es validada por los resultados de Bernal et al., que estudió la vía por la que las fibras zonulares anteriores y posteriores están conectadas a la cápsula de lente14. Usando el diseño de los zapatos personalizados que sólo requieren el lente, la zónula y cuerpo ciliar, se intentó estudiar biomecánica lente replicando alojamiento fisiológica.

<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

El objetivo de este protocolo es imitar la biomecánica fisiológica alojamiento de una manera costo-eficiente y práctica. Alojamiento se consigue mediante la contracción del cuerpo ciliar y la relajación de las fibras de la zónula, que da como resultado el engrosamiento de la lente necesaria para la visión cercana. Aquí, presentamos una novela, un método simple en el cual se replica alojamiento por tensar los zonules conectados a la cápsula de la lente a través de una camilla de lente manual (MLS). Este método controla el estiramiento radial mediante una lente cuando se somete a una fuerza constante y permite una comparación de acomodar los lentes, que se pueden estirar, no acomodar los lentes, que no puede ser estirada. Lo importante, las parejas de camilla a los zonules directamente y no a la esclera del ojo, así requiriendo solamente la lente, zonules y del cuerpo ciliar en lugar de la muestra del mundo entero. Esta diferencia puede disminuir significativamente el costo de adquirir lentes de donantes cadáver por aproximadamente 62% en comparación a adquirir un mundo entero.

Ojos de cerdo, una muestra común para estudiar la presbicia mediante lentes que se extiende de4,15, se obtuvieron (n = 10) de un matadero local y el presente Protocolo fue utilizado para observar la capacidad de alojamiento de las lentes. Figura 5 A muestra la comparación de la lente porcina antes y después de que se extiende a través de la MLS. Hubo un promedio 0.19 ± 0,07 mm au...

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

Simulación de la mecánica del alojamiento de la lente a través de una camilla de lente Manual

Presentamos un método eficiente de estudiar lente alojamiento utilizando una camilla manual de la lente. Los imitadores de protocolo fisiológico alojamiento tirando los zonules conectado alrededor de la cápsula de la lente, de tal modo, que estira la 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

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

6.7K vistas.  University of Maryland. Presentamos un método eficiente de estudiar lente alojamiento utilizando una camilla manual de la lente. Los imitadores de protocolo fisiológico alojamiento tirando los zonules conectado alrededor de la cápsula de la lente, de tal modo, que estira la lente.

Video: 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.

Estiramiento secuencias cortas de ADN con fuerza constante pinzas axial óptico

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

Investigación

Bioingeniería

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.

El estiramiento células micropatterned en una membrana de PDMS

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.

Una Plataforma Novela Estiramiento para Aplicaciones en Celular y Tisular Mecanobiología

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.

Imágenes de células vivas durante el estiramiento mecánico

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.

Síntesis de elastómeros suaves de polisiloxano-urea para aplicación de Lente Intraocular

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.

Producción de un dispositivo de medición de tensión con una impresora 3D mejorada

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

Ingeniería

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.

Cepa controlada de hidrogeles 3D bajo imágenes de microscopía en vivo

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

Caracterización Biomecánica de Lente Ocular Porcina con Compresión 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 destacado: Avanzando en la comprensión de los cambios en la rigidez del cristalino relacionados con la edad 

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

Prueba de compresión automatizada de la lente ocular

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

Simulación de la mecánica del alojamiento de la lente a través de una camilla de lente Manual

Resumen

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