Los autores desean dar las gracias a Elena Lomakina, Bauserman Richard Youngman Margaret, Vaknin Shay, Snyder Jessica, Striemer Chris, Nataraj Nakul, Hung Li Chung, Khire Tejas, y Eric Lam por su ayuda en este proyecto. Este proyecto fue financiado por el NIH PO1 HL # 018208.

1. Métodos 1,1 Celular Cámara de Flujo Una cámara de flujo, que se muestra en la Figura 1, fue construido de manera que las células pueden ser cultivadas bajo un esfuerzo cortante de 1,0 Pa (10 dinas / cm 2) y luego se transfiere directamente a un asilo MFP3D AFM (Santa Barbara, CA). <ol><li> La cámara de flujo se preparó para el experimento por primera limpieza de los portaobjetos de vidrio en solución Piranha (3:1 H 2 SO 4: H 2 O 2) durante 15 min y después de lavarlas con agua destilada. Ellos se cocieron luego a seco y recubierto con trietoxi aminopropil silano (APTES) en una cámara de deposición en vacío. </li><li> Una junta de silicona se cortan con una herramienta de corte Silhouette SD. Esto nos permitió controlar finamente las dimensiones de la cámara de flujo para el control de la velocidad de flujo y la tensión de cizallamiento durante el crecimiento celular. Típicamente, un canal se cortó 6,4 mm de ancho por 19 mm de largo a partir de una lámina de silicona 0,4 mm. El caudal necesario para géneroste una tensión de cizalladura de 1,0 Pa (10 dinas / cm 2) se calcula suponiendo que el flujo laminar en un canal rectangular con la ecuación: </li></ol><img alt="Ecuación 1" fo:content-width="0.737in" fo:src="/files/ftp_upload/50163/50163eq1highres.jpg" src="/files/ftp_upload/50163/50163eq1.jpg" /> donde Q es el caudal, τ es el esfuerzo cortante, μ es la viscosidad del medio, que aquí suponen 1,0 mPa (0,01 dyn * s / cm 2), h es la altura y w es la anchura de la cámara de flujo . <ol start="3"><li> La pieza superior de la cámara de flujo se alinea con la junta en la placa de cultivo celular y se fija con un anillo magnético. El conjunto se llenó con alcohol isopropílico (IPA) para la esterilización. </li><li> El sistema de flujo total fue montado. Los puertos de flujo en la placa de cultivo celular se conecta a las válvulas de tres vías. Las válvulas se conectaron a abrir 30 ml jeringas. IPA se eliminan a través del sistema, que después se lavó con 30 ml de medio de McCoy con 4% de suero fetal de ternera (FCS). El sistema se llenó entonces con 20 ml del medio de Vec Tecnologías de crecimiento celular. Las cubiertas fueron colocados en la parte superior de las jeringas. El depósito de captura tapa de la jeringa tenía una aguja en su lugar para mover medio de vuelta al depósito de alimentación. Estéril de 0,2 micras filtros se fijaron a las entradas de aire en las cubiertas para evitar la contaminación del sistema. La cámara de flujo estaba entonces listo para la siembra de células. </li></ol> 1,2 Cultivo Celular <ol><li> Humanos de las células endoteliales de vena umbilical humana (HUVEC) y el medio de crecimiento se compraron Tecnologías de Vec (Rensselaer, NY) y se cultivaron hasta confluencia en un matraz T25. </li><li> El medio de crecimiento se retiró del matraz y la monocapa de células liberadas con 2 ml de tripsina al 2,5%. Una vez que las células estaban en solución, la tripsinización se detuvo con la adición de 10 ml de medio de cultivo celular al matraz. </li><li> Thsuspensión e celular se centrifugó durante 5 min y se eliminó el sobrenadante. Las células se resuspendieron en 1 ml de medio de cultivo celular (que contiene suero) para la inyección en la cámara de flujo. </li><li> La suspensión de células (0,5 ml, ~ 50.000 células) se cargó en una jeringa y se inyecta en la cámara de flujo a través de una válvula de tres vías. </li><li> Las células se dejaron sedimentar y se adhieren al sustrato de vidrio durante 2 horas antes de flujo se inició. Las células fueron cultivadas bajo flujo en una incubadora a 37 ° C durante 1 a 5 días hasta confluencia. </li></ol> 1,3 Cantilever Preparación y sangría Celular <ol><li> Sin punta de AFM (voladizos nanomundo, Suiza) se limpiaron en ácido nítrico durante 5 min y se funcionaliza con trietoxi aminopropil silano (APTES) en una cámara de deposición de vapor. </li><li> Una solución de 5 mg / ml en peso NHS-sulfo-LC-biotina en solución salina tamponada de Hank (HBSS) se preparó. Los voladizos se sumergieron en la solución durante 15 min a conjula puerta del silano con N-hidroxisuccinimida (NHS) química. </li><li> Una solución de medio de biotina libre se realizó mediante la incubación de 20 ml de medio de cultivo Vec Cell Technologies (incluido el suero) con 200 l de perlas de estreptavidina durante 12 h. Las perlas se retiraron del medio con un imán y el medio se filtró a través de un filtro estéril de 0,22 micras. </li><li> La cámara de flujo fue retirado de la placa de cultivo celular y las células se lavaron en 37 ° C biotina libre de soporte. </li><li> Una solución stock de 1 l de 2,4 micras perlas recubiertas con estreptavidina en 1 ml de medio libre de biotina fue preparado, y 100 l de la madre se añadió a la placa de cultivo celular. </li><li> Perlas de estreptavidina fueron recogidos con el voladizo por el aterrizaje de la punta en la superficie de vidrio junto a una retracción de bolas, posicionando el vértice de la viga en voladizo sobre el cordón, y después presionando el voladizo hacia abajo sobre los varios segundos de talón y de descanso para. </li><li> La sensibilidad de la medida era voladizod por el sangrado en una región de vidrio desnudo y utilizando la pendiente de la curva para ajustar la desviación de la punta como una función de la tensión. </li><li> La constante de resorte de la viga en voladizo se calculó a continuación a partir de una calibración térmica en el software MFP3D. </li><li> El voladizo calibrado se utilizó a continuación para sangrar las muestras, como se muestra en la Figura 2. Estas perlas de 2,4 micras ofrecer una mayor área de contacto con la superficie de la célula de modo que las propiedades mecánicas de la capa blanda glicocálix puede ser detectado. El voladizo se coloca sobre una celda cerca del núcleo de la célula y un enfoque suave de la punta en la celda se usa para ajustar la altura del voladizo aproximadamente 3 m por encima de la superficie de la célula. El software se fijó para 20 muescas repetidos a una velocidad de 1 m / seg a una fuerza máxima de 7 nN. Aproximadamente 6 segundos transcurrido entre los contactos sucesivos. Es posible utilizar diferentes tasas de sangría para la prueba de tiempo que dependen de las propiedades del glicocálix, aunque en estos inicial<html> experimentos, sólo una tasa de indentación simple (1 m / seg) se utilizó. </li></ol> 2. Sangría Theory Indentación en un semiespacio elástico con una esfera de radio R puede ser descrito usando la teoría de Hertz, donde se da la fuerza de indentación, F, por la ecuación: <img alt="Ecuación 2" fo:content-width="1.157in" fo:src="/files/ftp_upload/50163/50163eq2highres.jpg" src="/files/ftp_upload/50163/50163eq2.jpg" /> Donde δ es la profundidad de penetración y * E es el módulo reducida del material bajo prueba (Figura 3). En el caso de un indentador infinitamente rígido uniforme que incide un semiespacio elástico, E * viene dada por la ecuación: 3/50163eq3.jpg &quot;/&gt; donde E es el módulo de elasticidad y ν es el coeficiente de Poisson del material. El trabajo reciente con películas de polímero ha inspirado el desarrollo de un modelo de dos capas para la determinación del módulo de elasticidad y espesor de las películas delgadas 1. Estamos aplicando este modelo a la biología celular mediante el tratamiento de la glicocálix como una película uniforme suave delgada sobre la superficie del cuerpo de la célula. Usando este modelo, el módulo reducida del sistema se convierte en: <img alt="Ecuación 4" fo:content-width="2.85in" fo:src="/files/ftp_upload/50163/50163eq4highres.jpg" src="/files/ftp_upload/50163/50163eq4.jpg" /> Donde E es el módulo de GC del glicocálix, célula E es el módulo de la célula cuerpo, P, Q y n son constantes que han sido determinados empíricamente a partir del polímero se ajusta, y z viene dada por la ecuación: <p class="jove_content" fo:keep-Together.within-page = &quot;always&quot;&gt; <img alt="Ecuación 5" fo:content-width="1.257in" fo:src="/files/ftp_upload/50163/50163eq5highres.jpg" src="/files/ftp_upload/50163/50163eq5.jpg" /> Donde t es el espesor de la capa de glicocálix. Un esquema de estos parámetros se muestra en la Figura 3. El modelo ha demostrado ser una forma precisa de determinar el módulo y el espesor de una película delgada sobre el agravamiento de sustrato 1. Esta ecuación se puede utilizar para adaptarse a las curvas obtenidas a partir de indentación en las células para determinar el módulo y el espesor de la glicocálix endotelial, como se muestra en la Figura 4.

<ol>
	<li>Clifford, C., Seah, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoindentation+measurement+of+young's+modulus+for+compliant+layers+on+stiffer+substrates+including+the+effect+of+poisson's+ratios.">Nanoindentation measurement of young's modulus for compliant layers on stiffer substrates including the effect of poisson's ratios.</a> Nanotechnology. , (2009).</li><li>Gouverneur, M., Spaan, J. A. E., Pannekoek, H., Fontijn, R. D., Vink, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+shear+stress+stimulates+incorporation+of+hyaluronan+into+endothelial+cell+glycocalyx.">Fluid shear stress stimulates incorporation of hyaluronan into endothelial cell glycocalyx.</a> Am. J. Physiol. Heart. Circ. Physiol. 290 (1), 458-452 (2006).</li><li>Hocde, S. A., Hyrien, O., Waugh, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+molecule+distribution+relative+to+neutrophil+surface+topography+assessed+by+tirfm.">Cell adhesion molecule distribution relative to neutrophil surface topography assessed by tirfm.</a> Biophysical Journal. 97 (1), 379-387 (2009).</li><li>Lipowski, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+glycocalyx+as+a+barrier+to+leukocyte+adhesion+and+its+mediation+by+extracellular+proteases.">The endothelial glycocalyx as a barrier to leukocyte adhesion and its mediation by extracellular proteases.</a> Annals of biomedical engineering. 40 (4), 840-848 (2012).</li><li>Lu, L., Oswald, S. J., Ngu, H., Yin, F. C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+actin+stress+fibers+in+living+cells.">Mechanical properties of actin stress fibers in living cells.</a> Biophysical Journal. 95 (12), 6060-6071 (2008).</li><li>Pries, A. R., Secomb, T. W., Gaehtgens, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+surface+layer.">The endothelial surface layer.</a> Pflugers Archiv. European Journal of Physiology. 440 (5), 653-666 (2000).</li><li>Spillmann, C. M., Lomakina, E., Waugh, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+adhesive+contact+dependence+on+impingement+force.">Neutrophil adhesive contact dependence on impingement force.</a> Biophysical Journal. 87 (6), 4237-4245 (2004).</li><li>vanden Berg, B. M., Vink, H., Spaan, J. A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+glycocalyx+protects+against+myocardial+edema.">The endothelial glycocalyx protects against myocardial edema.</a> Circulation Research. 92 (6), 592-594 (2003).</li><li>Williams, T. E., Nagarajan, S., Selvaraj, P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+impact+of+membrane+microtopology+on+effective+two-dimensional+affinity.">Quantifying the impact of membrane microtopology on effective two-dimensional affinity.</a> J. Biol. Chem. 276 (16), 13283-138 (2001).</li><li>Vink, H., Duling, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Distinct+Luminal+Domains+for+Macromolecules,+Erythrocytes,+and+Leukocytes+Within+Mammalian+Capillaries.">Identification of Distinct Luminal Domains for Macromolecules, Erythrocytes, and Leukocytes Within Mammalian Capillaries.</a> Circulation Research. 79, 581-589 (1996).</li></ol>

No hay conflictos de interés declarado.

Se utilizaron los valores calculados a partir del modelo de dos capas y la teoría de Hertz para modelar la interacción de un leucocito circulante en la sangre con la pared endotelial. Hemos calculado que un microvellosidades en el recuento de leucocitos con un diámetro de 50 nm bajo una carga de 10 pN guión sería de aproximadamente 150 nm en el glucocáliz, sólo una fracción del espesor total. Esto indica que el glucocáliz, con propiedades como medidos en este experimento, es una barrera significativa para la in...

<table border="1"><tbody><tr><td> Nombre del reactivo / material </td><td> Empresa </td><td> Número de Catálogo </td><td> Comentarios </td></tr><tr><td> Medio de McCoy </td><td> Gibco </td><td> 16600-082 </td><td></td></tr><tr><td> Suero de ternero fetal </td><td> Hyclone </td><td> SH30070 </td><td></td></tr><tr><td> La célula endotelial Medio de Crecimiento </td><td> Vec Tecnologías </td><td> MCDB-131 </td><td></td></tr><tr><td> Humano agrupado Las células endoteliales de vena umbilical </td><td> Vec Tecnologías </td><td> PHUVEC/T-25 </td><td></td></tr><tr><td> Ácido sulfúrico </td><td> JT Baker </td><td> 9681-02 </td><td></td></tr><tr><td> Peróxido de hidrógeno </td><td> VWR </td><td> BDH3742-1 </td><td></td></tr><tr><td> (3-aminopropil) trietoxisilano </td><td> Aldrich </td><td> 440140-100ML </td><td></td></tr><tr><td> Alcohol isopropílico </td><td> VWR </td><td> BDH8999-4 </td><td></td></tr><tr><td> Tripsina </td><td> Cellgro </td><td> 25-054 C1- </td><td></td></tr><tr><td> Tamponada de Hank Solución salina </td><td> Gibco </td><td> 14175-095 </td><td></td></tr><tr><td> sulfo-NHS-LC-biotina </td><td> Thermo Scientific </td><td> 21335 </td><td></td></tr><tr><td> Estreptavidina cuentas </td><td> Dynabeads </td><td> 112.06D </td><td></td></tr><tr><td> MFP-3D AFM </td><td> Asylum Research </td><td></td><td></td></tr><tr><td> Cantilevers sin punta </td><td> Nanomundo </td><td> ARROW-TL1-50 </td><td></td></tr><tr><td> Silhouette SD </td><td> Quickutz </td><td> Silueta-SD </td><td></td></tr><tr><td> Caucho de silicona </td><td> Stockwell Elastomerics </td><td> SE50-RS </td><td></td></tr><tr><td> 30 ml Jeringas </td&gt; <td> Benton Dickinson </td><td> 309650 </td><td></td></tr><tr><td> 18 agujas de calibre </td><td> Benton Dickinson </td><td> 305196 </td><td></td></tr><tr><td> Sets de extensión </td><td> Hospira </td><td> 4429-48 </td><td></td></tr><tr><td> Válvulas de 4 vías </td><td> Teleflex </td><td> W21372 </td><td></td></tr><tr><td> Hombre / Mujer Caps puerto </td><td> Smith Medical </td><td> MX491B </td><td></td></tr><tr><td> Bomba peristáltica </td><td> Watson-Marlow </td><td> 401U / D </td><td></td></tr><tr><td> Tubería peristáltica </td><td> Watson-Marlow </td><td> 903.0016.016 </td><td></td></tr><tr><td> filtros estériles </td><td> Pall Life Science </td><td> 4652 </td><td></td></tr></tbody></table>

quantifying the mechanical properties of the endothelial glycocalyx with atomic force microscopy

Nuestro entendimiento de la interacción de los leucocitos y la pared del vaso durante la captura de leucocitos está limitado por una comprensión incompleta de las propiedades mecánicas de la capa de la superficie endotelial. Se sabe que las moléculas de adhesión en los leucocitos se distribuyen de manera no uniforme en relación con topografía de la superficie 3, que limita la formación de topografía unión adhesiva con otras superficies 9, y que las fuerzas de contacto fisiológicas (≈ 5,0 a 10,0 pN por microvellosidades) puede comprimir las microvellosidades como poco como un tercio de su longitud en reposo, el aumento de la accesibilidad de las moléculas a la superficie opuesta 3, 7. Consideramos el endotelio como una estructura de dos capas, el cuerpo celular relativamente rígido, además de la glucocáliz, un recubrimiento de azúcar blando protectora en la superficie luminal 6. Se ha demostrado que el glicocáliz puede actuar como una barrera para reducir la adhesión de los leucocitos a la superficie endotelial 4.En este informe se empiezan a abordar la deformabilidad de las superficies endoteliales para entender cómo la rigidez mecánica del endotelio puede afectar la formación del enlace. Las células endoteliales cultivadas en cultivo estático no expresan un glicocalix robusta, pero las células cultivadas bajo condiciones de flujo fisiológico comienzan a aproximar el glucocáliz observado in vivo 2. El módulo del cuerpo de células endoteliales ha sido medida mediante microscopía de fuerza atómica (AFM) para ser aproximadamente de 5 a 20 kPa 5. El espesor y la estructura del glicocálix se han estudiado utilizando microscopía electrónica de 8, y el módulo de la glicocálix se ha aproximado mediante métodos indirectos, pero a nuestro conocimiento, no se han publicado informes de una medición directa del módulo glicocálix en células vivas . En este estudio, se presentan experimentos de indentación realizadas con una sonda AFM novela en células que han sido cultivadas en condiciones para maximizar su expresión glicocálix a make mediciones directas del módulo y el espesor de la glicocálix endotelial.

En un experimento típico, 20 fuerza-distancia-vs curvas se obtuvieron a partir de una región determinada de la célula, típicamente en la región perinuclear, cerca, pero no en, el núcleo (a menos de ~ 2 micras). Las curvas se ajustaron para tener en cuenta cualquier deriva de la muestra durante toda la duración de la medición y se promediaron para eliminar el ruido en voladizo, como se muestra en la Figura 4. Las curvas se analizaron y encajar con el modelo de dos capas que fue desarrollado para ...

Watch this Scientific Journal Video about Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy at JoVE.com

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Las características mecánicas de glicocálix endotelial se midieron por indentación usando esferas micrón en voladizo de AFM. Las células endoteliales se cultivaron en una cámara de costumbre bajo condiciones de flujo fisiológico para inducir la expresión glicocálix. Los datos se analizaron utilizando un modelo de película delgada para determinar el espesor y el módulo de glicocálix.

La cuantificación de las propiedades mecánicas de la glicocálix endotelial con la Microscopía de Fuerza Atómica

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

quantifying-mechanical-properties-endothelial-glycocalyx-with-atomic

Research

JoVE Journal

Bioengineering

13.1K vistas.  University of Rochester. Las características mecánicas de glicocálix endotelial se midieron por indentación usando esferas micrón en voladizo de AFM. Las células endoteliales se cultivaron en una cámara de costumbre bajo condiciones de flujo fisiológico para inducir la expresión glicocálix. Los datos se analizaron utilizando un modelo de película delgada para determinar el espesor y el módulo de glicocálix.

Video: Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

Inmovilización de bacterias para tratamiento de imágenes de microscopía de fuerza atómica

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Investigación

Bioingeniería

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1) 1,2. Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. 
The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation 3,such as Ni2+ or Mg2+, which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase 4 and a repair enzyme 5, bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GR-chaperone protein complexes 1,2 and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.

A tapping mode atomic force microscope (AFM) method for the visualization of plasmid DNA, cytoplasmic proteins, and DNA-protein complexes is described. The method includes alternate approaches for preparing samples for AFM imaging following biochemical manipulation. DNA containing specific protein interacting regions are observed in near-physiologic buffer conditions.

La visualización de ADN recombinante y complejos de proteínas utilizando microscopía de fuerza atómica

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Medición de las propiedades mecánicas de las células vivas mediante microscopía de fuerza atómica

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

Un nuevo método para la localización de cuentas fluorescentes Reporter cerca de la superficie de la célula de la cultura para Microscopía de Fuerza de Tracción

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

Fuerza Atómica Microscopía Imaging y Fuerza Espectroscopia de admitidos Bilayers lípidos

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

En Mapping in situ de las propiedades mecánicas de biopelículas por partículas de seguimiento Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

Síntesis de hidrogeles con propiedades antiincrustantes como membranas de purificación de agua

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

A Millimeter Scale Flexural Testing System for Measuring the Mechanical Properties of Marine Sponge Spicules

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the flexural behavior of these LBBSs is important for understanding the origins of their remarkable mechanical functions.
We describe a protocol for performing three-point bending tests using a custom-built mechanical testing device that can measure forces ranging from 10-5 to 101 N and displacements ranging from 10-7 to 10-2 m. The primary advantage of this mechanical testing device is that the force and displacement capacities can be easily adjusted for different LBBSs. The device&#39;s operating principle is similar to that of an atomic force microscope. Namely, force is applied to the LBBS by a load point that is attached to the end of a cantilever. The load point displacement is measured by a fiber optic displacement sensor and converted into a force using the measured cantilever stiffness. The device&#39;s force range can be adjusted by using cantilevers of different stiffnesses.
The device&#39;s capabilities are demonstrated by performing three-point bending tests on the skeletal elements of the marine sponge Euplectella aspergillum. The skeletal elements&#8212;known as spicules&#8212;are silica fibers that are approximately 50 &#181;m in diameter. We describe the procedures for calibrating the mechanical testing device, mounting the spicules on a three-point bending fixture with a &#8776;1.3 mm span, and performing a bending test. The force applied to the spicule and its deflection at the location of the applied force are measured.

We present a protocol for performing three-point bending tests on sub-millimeter scale fibers using a custom-built mechanical testing device. The device can measure forces ranging from 20 &#181;N up to 10 N and can therefore accommodate a variety of fiber sizes.

Un milímetro a la flexión prueba sistema de la escala para medir las propiedades mecánicas de espículas de esponja marina

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive models, which provide a mathematical representation of the mechanical function of those structures. This presented biaxial mechanical testing protocol involves (i) tissue acquisition, (ii) the preparation of tissue specimens, (iii) biaxial mechanical testing, and (iv) postprocessing of the acquired data. First, tissue acquisition requires obtaining porcine or ovine hearts from a local Food and Drug Administration-approved abattoir for later dissection to retrieve the valve leaflets. Second, tissue preparation requires using tissue specimen cutters on the leaflet tissue to extract a clear zone for testing. Third, biaxial mechanical testing of the leaflet specimen requires the use of a commercial biaxial mechanical tester, which consists of force-controlled, displacement-controlled, and stress-relaxation testing protocols to characterize the leaflet tissue&#39;s mechanical properties. Finally, post-processing requires the use of data image correlation techniques and force and displacement readings to summarize the tissue&#39;s mechanical behaviors in response to external loading. In general, results from biaxial testing demonstrate that the leaflet tissues yield a nonlinear, anisotropic mechanical response. The presented biaxial testing procedure is advantageous to other methods since the method presented here allows for a more comprehensive characterization of the valve leaflet tissue under one unified testing scheme, as opposed to separate testing protocols on different tissue specimens. The proposed testing method has its limitations in that shear stress is potentially present in the tissue sample. However, any potential shear is presumed negligible.

This protocol involves characterizations of atrioventricular valve leaflets with force-controlled, displacement-controlled, and stress-relaxation biaxial mechanical testing procedures. Results acquired with this protocol can be used for constitutive model development to simulate the mechanical behavior of functioning valves under a finite element simulation framework.

Biaxiales caracterizaciones mecánicas de válvulas auriculoventricular del corazón

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive ...

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

Aplicación de microscopía de fuerza atómica para detectar la osteoartritis temprana

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...