I protocolli seguenti sono accettati sotto l'Università del Maryland istituzionale Animal Care e Comitato di uso, nonché l'Institutional Review Board. I protocolli di seguono le norme federali, statali e locali, e gli orientamenti stabiliti dalla politica dell'Università del Maryland sulla biosicurezza.
1. dissezione del campione di occhio
<ol><li>
Ottenere un campione di occhio dalla banca locale del macello o del tessuto. Se si ottiene un globo intero occhio, estrarre immediatamente la lente, annesso zonules e vitroso. Nota: I dettagli specifici descritti di seguito si riferiscono agli occhi sia suini e umani.<ol>
<li>Utilizzando le pinze e le forbici chirurgiche disinfettate, tagliare e rimuovere tutto il tessuto in eccesso che circondano la sclera.</li>
		<li>Tenere saldamente l'occhio sul suo lato e, utilizzando una lametta, praticare una piccola incisione lungo il lato dell'occhio 3 mm dalla cornea. Fare il taglio profondo abbastanza per aver raggiunto il vitreo all'interno dell'occhio.</li>
		<li>Utilizzando le forbici, tagliare con cura ulteriormente lungo l'incisione attorno alla circonferenza dell'occhio. Evitare di perforare la lente. Un'immagine rappresentativa è illustrata nella Figura 2A.</li>
		<li>Una volta che la circonferenza esterna dell'occhio è stata tagliata, rimuovere il tessuto posteriore dell'occhio usando il forcipe. Isolare la lente, zonules, corpo ciliare e il vitroso allegato con il forcipe. Un'immagine rappresentativa è illustrata nella Figura 2B.</li>
		<li>Usando le forbici e pinze, rimuovere vitroso in eccesso in modo che la lente può porre piatto sul MLS. Nota: In caso di trapianto di cornea, il tasto corneale viene utilizzato in chirurgia e il resto del globo è disponibile per scopi di ricerca. Tuttavia, questo globo parziale ancora utilizzabile nella preparazione dei tessuti dell'apparato di barella lente. Se solo la parte posteriore è ottenuto, eseguire solo il passaggio 1.1.4–1.1.5.</li>
	</ol>
</li>
	<li>Disinfettare tutte le post-dissezione attrezzature usate in soluzione di candeggina al 15% per 30 min.</li>
</ol>2. prova di montaggio della barella lente manuale
<ol><li>Inserire i fondi di scarpa di 10 mm e il corrispondente scarpa top nella piastra inferiore della MLS così ci rimane un gap di 5 mm tra la parete posteriore del trattino scarpa e scarpa stessa.</li>
	<li>Allineare le piastre superiore e inferiore, scattare le piastre insieme; il dispositivo è ora in posizione di distenderla.</li>
	<li>Inserire le piastre nel caso la piastra e la vite di arresto nel foro situato sul lato della piastra di fondo.</li>
	<li>Inserire il caso di piastra base e inserire la chiave i trattini allineati.</li>
	<li>Ruotare la chiave in senso orario fino a raggiungere la vite di arresto per contratto le scarpe e torsione indietro in senso antiorario per restituire le scarpe nella posizione originale non stirata.</li>
</ol>3. montaggio della lente
<ol><li>Inserire il fondo scarpa 10 mm la piastra inferiore nella MLS affinché una 5 mm distanza tra rimane la parete posteriore del trattino la scarpa e la scarpa stessa.</li>
	<li>Usando il forcipe curvo, posizionare estratti lente a faccia in su al centro della piastra inferiore in modo che le scarpe stanno sostenendo la lente sopra il foro centrale.</li>
	<li>Incastrarsi parte superiore corrispondente delle scarpe, ritaglio solo gli zonules e vitroso. Verificare visivamente che la lente rimane come centrato possibile sulla piastra inferiore.</li>
	<li>Ripetere i passaggi da 2.3 – 2.4.</li>
</ol>4. misurazione della lente
<ol><li>Posizionare un sistema di imaging direttamente sopra l'apparato al fine di catturare video e immagini del processo d'allungamento. Assicurarsi di includere un righello nella cornice dell'immagine con precisione dimensioni e adattare le immagini in post-elaborazione. Nota: Qualsiasi sistema di imaging adatto è sufficiente per questo passaggio; Qui usiamo un 12 megapixel, messa a fuoco automatica smartphone un piede dal campione.</li>
	<li>Con fermezza ma senza intoppi, ruotare la chiave in senso orario per allungare la lente. La figura 3 Mostra immagini rappresentative allo stato distenderla e allungato.</li>
	<li>Dopo aver fotografato la lente allungata, ruotare la chiave in senso antiorario per ripristinare il campione al suo stato di riposo. Nota: È indispensabile che la misura della lente viene eseguita in modo tempestivo al fine di ridurre al minimo la disidratazione della lente.</li>
	<li>Chiaramente fotografare lo stato di riposo finale della lente.</li>
</ol>5. analisi dei dati
<ol><li>Caricare l'immagine di ImageJ e, utilizzare la funzionalità di "punto" per selezionare almeno 40 punti sulla circonferenza della lente come mostrato in Figura 4A. Utilizzare l'opzione di "Misurare" → "Analizza" per ottenere la posizione di ogni punto selezionato.</li>
	<li>Va bene (utilizzando software ad esempio MATLAB) i punti di posizione al fine di produrre un raggio e chi-quadrato del fit come mostrato in Figura 4B. Convertire il raggio dei pixel e l'errore in metriche utilizzando il righello fotografato.</li>
	<li>Eseguire due munito test t accoppiato al confronto una singola lente prima e dopo lo stretching 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 ha interesse di proprietà in Bioniko Consulting LLC.

Abbiamo messo a punto un nuovo metodo per fornire un accurato ed efficiente modo di studiare la capacità di alloggio dell'obiettivo utilizzando un meccanismo di bloccaggio dual-pezzo di accoppiare la barella al campione. Durante il soggiorno, si distende la lente e il diametro diminuisce in risposta a rilassamento del fibre zonular1,2,4,19. Il metodo si concentra su questo fenomeno di bloccagg...

Alloggio è il processo mediante il quale l'occhio umano è in grado di regolare dinamicamente la forma del suo cristallino per vedere gli oggetti a distanze lontane o vicine a fuoco. Alloggio è un processo intrinsecamente biomeccanico. Su stimolo neurale, muscoli ciliari producono una forza sul corpo ciliare e per le fibre di zonule che applicare alla circonferenza della capsula di obiettivo1,2. Mentre ci sono diverse teorie dietro la biomeccanica di alloggi, il più ampiamente accettata è l'ipotesi di Helmholtz. Secondo l'ipotesi, la lente è in uno stato naturale allungato, corrispondente alla forma più sottile della lente che è ottimale per la messa a fuoco di oggetti distanti. Per cambiare messa a fuoco oggetti vicini, i muscoli ciliari si contraggono e si distendono le fibre zonulare. A sua volta, l'obiettivo si addensa, aumentando le curvature di superficie anteriore e posteriore. Questo corrisponde ad un aumento nel potere diottrico che è necessario per la visione, di conseguenza, una più breve lunghezza focale1da vicino.
La capacità di ospitare è compromessa nel tempo tramite una condizione denominata presbiopia. Interessa tutti entro i 50 anni, la presbiopia rende l'occhio in grado di modificare dinamicamente la messa a fuoco da lontano per chiudere distanze3. Per combattere la presbiopia, gli attuali metodi sono passivi tra cui lenti bifocali e lenti correttive. Aumentando la capacità di concentrarsi su oggetti vicini a alcuni aerei, tali trattamenti passivi non possono ripristinare la capacità di fuoco dinamico della lente4,5. Al fine di trattare in modo efficiente la presbiopia, o possibilmente prevenirlo, c'è un bisogno costante di comprendere meglio alloggi.
Per studiare lens alloggio, un numero di dispositivi è stato sviluppato per simulare il fenomeno ex vivo4,6,7,8,9. Dischi di filatura sono stati introdotti per monitorare l'allungamento della lente via forze centrifughe8. Per replicare più fedelmente il fenomeno, dispositivi di stretching lente sono stati gradualmente introdotti e innovati. Utilizzando una lente barella, Manns et al. caratterizzato la forza necessaria per accogliere la lente mentre correlando tale obiettivo potenza e diametro equatoriale9. Comprensione corrente è che la lente si irrigidisce con l'età, con conseguente cambiamento ridotto a forma di lente in risposta ad una forza uguale dal corpo ciliare3,10,11,12.
Corrente lente barelle spesso comportano un'installazione complessa, attuazione elettronica e programmabili tassi d'allungamento e richiede il cadavere intero bulbo oculare6,7,10,13. Questo requisito aumenta il costo per esperimento a oltre $500.00 per l'occhio e diminuisce la disponibilità del campione. Qui presentiamo un metodo per replicare lens alloggio a basso costo come l'occhio posteriore ammonta a circa $200,00. Mentre meno sofisticato rispetto a molti dispositivi utilizzati oggi, la tecnica è molto più redditizio e adottabile senza compromettere i risultati. Questo metodo è centrato intorno una barella di lente manuale (MLS), raffigurata in Figura 1e utilizza un esclusivo sistema di bloccaggio sulle fibre zonulare e un metodo di torsione radiale per espandere il diametro della lente. La precisione fisiologica del protocollo viene convalidata dai risultati di Bernal et al., che ha studiato il percorso attraverso il quale le fibre zonulare anteriori e posteriori sono collegate al capsula lente14. Utilizzando il design di scarpe personalizzate che richiedono solo la lente, zonule e corpo ciliare, abbiamo mirato a studiare lente biomeccanica replicando alloggi fisiologico.

<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

L'obiettivo del presente protocollo è quello di imitare la biomeccanica del fisiologico alloggio in maniera efficiente, pratico. Alloggio avviene attraverso la contrazione del corpo ciliare e rilassamento delle fibre zonule, che provoca l'ispessimento della lente necessaria per la visione da vicino. Qui, presentiamo un romanzo, un semplice metodo in cui alloggio è replicato da tensing zonules collegato alla capsula del cristallino tramite una barella di lente manuale (MLS). Questo metodo controlla l'allungamento radiale raggiunto da una lente quando sottoposti a una forza coerenza e consente un confronto di accogliere lenti, che possono essere allungati, a lenti non accomodante, che non possono essere allungati. D'importanza, le coppie di barella il zonules direttamente e non la sclera dell'occhio, così richiedendo solo la lente, zonules e corpo ciliare anziché il campione di tutto il globo. Questa differenza può ridurre notevolmente il costo di acquisto di lenti di donatore cadavere di circa 62% rispetto all'acquisizione di un intero globo.

Porcini occhi, un campione comune per lo studio della presbiopia tramite lente stretching4,15, sono stati ottenuti, (n = 10) da un mattatoio e il presente protocollo è stato utilizzato per osservare la capacità di alloggio delle lenti. Figura 5 A Mostra il confronto della lente porcino prima e dopo l'allungamento tramite MLS. C'era un aumento media 0,19 ± 0,07 mm raggio di lente qu...

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 la meccanica del Lens Alloggio tramite una barella lente manuale

Presentiamo un metodo efficiente per studiare lens alloggio utilizzando una barella lente manuale. Il protocollo imita fisiologico alloggi tirando il zonules collegato intorno alla capsula di obiettivo, quindi, che allunga 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

Research

JoVE Journal

Bioengineering

6.7K Views.  University of Maryland. Presentiamo un metodo efficiente per studiare lens alloggio utilizzando una barella lente manuale. Il protocollo imita fisiologico alloggi tirando il zonules collegato intorno alla capsula di obiettivo, quindi, che allunga 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.

Stretching brevi sequenze di DNA con pinzette costante forza assiale ottici

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

Ricerca

Bioingegneria

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.

Stretching celle micropatterned su una membrana 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 piattaforma Novel Stretching per applicazioni in cellule e tessuti 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.

Imaging Live Cell durante meccanica Stretch

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.

Sintesi degli elastomeri molli polisilossano-urea per applicazione di lente intraoculare

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

Chimica

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.

Produzione di un dispositivo di misurazione della deformazione con una stampante 3D migliorata

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

Ingegneria

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.

Ceppo controllato di idrogel 3D in imaging a microscopia dal vivo

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

Caratterizzazione biomeccanica del cristallino oculare suino con compressione automatizzata

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.

Autore in primo piano: Migliorare la comprensione dei cambiamenti di rigidità delle lenti legati all'età 

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

Test di compressione automatizzato della lente oculare

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

Simulando la meccanica del Lens Alloggio tramite una barella lente manuale

Riepilogo

Esplora Altri Video

PLAYLIST