Os autores gostariam de agradecer a Elena Lomakina, Richard Bauserman, Margaret Youngman, Shay Vaknin, Jessica Snyder, Chris Striemer, Nakul Nataraj, Hung Li Chung, Tejas Khire, e Eric Lam por sua ajuda com este projeto. Este projeto foi financiado pelo NIH # PO1 HL 018208.

1. Métodos Câmara de caudal 1,1 Célula A câmara de fluxo, mostrado na Figura 1, foi construído de modo a que as células podem ser cultivadas sob um corte de 1,0 Pa (10 dyn / cm 2) e, em seguida, transferido directamente para uma Asylum MFP3D AFM (Santa Barbara, CA). <ol><li> A câmara de fluxo foi preparado para o ensaio, em primeiro lugar a limpeza das lâminas de vidro, em solução piranha (3:1 H 2 SO 4: H 2 O 2) durante 15 min e, em seguida, lavando-as com água destilada. Eles foram então cozidos a seco e revestidos com aminopropil-trietoxi-silano (APTES), numa câmara de deposição de vácuo. </li><li> A junta de silicone foi cortada utilizando uma ferramenta de corte Silhouette SD. Isto permitiu-nos controlar com precisão as dimensões de fluxo da câmara de controlo da taxa de fluxo e tensão de cisalhamento durante o crescimento celular. Tipicamente, um canal foi cortado 6,4 milímetros de largura por 19 mm de comprimento a partir de uma folha de silicone de 0,4 mm. A vazão necessária para gêneroste uma tensão de corte de 1,0 Pa (10 dines / cm 2) é calculada assumindo um fluxo laminar de um canal rectangular com a equação: </li></ol><img alt="Equação 1" fo:content-width="0.737in" fo:src="/files/ftp_upload/50163/50163eq1highres.jpg" src="/files/ftp_upload/50163/50163eq1.jpg" /> onde Q é a taxa de fluxo, τ é a tensão de cisalhamento, μ é a viscosidade do meio, aqui assumido como sendo 1,0 mPa (0,01 dines * s / cm 2), h é a altura e w é a largura da câmara de fluxo . <ol start="3"><li> A parte superior da câmara de fluxo alinhado com o vedante na placa de cultura de células e fixado com um anel magnético. O conjunto foi cheio com álcool isopropílico (IPA) para a esterilização. </li><li> O sistema de escoamento total foi montado. As portas de fluxo na placa de cultura de células foram conectados a válvulas de três vias. As válvulas foram conectados a abrir 30 ml seringas. IPA foi borbulhada através do sistema, o qual foi então lavado com 30 ml de meio de McCoy com 4% de soro fetal bovino (FCS). O sistema foi então cheio com 20 ml do meio de crescimento de células Vec Technologies. As tampas foram colocadas sobre as partes superiores das seringas. A captura do reservatório tampão tinha uma seringa de agulha no lugar para mover meio de volta para o reservatório de alimentação. Estéreis filtros de 0,2 mM foram ligados às entradas de ar nas tampas para evitar a contaminação do sistema. A câmara de fluxo foi então pronto para a sementeira de células. </li></ol> 1,2 Cultura de Células <ol><li> As células endoteliais da veia umbilical humana (HUVEC) quer do meio de cultura foram adquiridos a partir de Vec Technologies (Rensselaer, NY) e crescidas até à confluência num balão T25. </li><li> O meio de crescimento foi removido do balão e a monocamada de células libertadas com 2 ml de tripsina 2,5%. Uma vez que as células estavam em solução, a tripsinização foi interrompida com a adição de 10 ml de meio de cultura de células para o balão. </li><li> Thsuspensão de células e centrifugou-se durante 5 min e o sobrenadante foi removido. As células foram ressuspensas em 1 ml de meio de cultura celular (contendo soro) para injecção no interior da câmara de fluxo. </li><li> A suspensão de células (0,5 ml, ~ 50.000 células) foi carregada para uma seringa e injectado na câmara de fluxo através de uma válvula de três vias. </li><li> As células foram deixadas assentar e aderir ao substrato de vidro por 2 horas antes de o fluxo foi iniciado. As células foram cultivadas sob fluxo dentro de uma incubadora a 37 ° C durante 1 a 5 dias até à confluência. </li></ol> 1,3 Preparação Cantilever e recuo celular <ol><li> Cantilever sem cantilevers de AFM (NanoWorld, Suíça) foram limpas em ácido nítrico durante 5 min e funcionalizado com aminopropil-trietoxi-silano (APTES), numa câmara de deposição de vapor. </li><li> Uma solução de 5 mg / ml, em peso, NHS-sulfo-LC-biotina, em solução salina tamponada de Hank (HBSS) foi preparado. As vigas foram submergidos na solução durante 15 min a conjuportão do silano com N-hidroxi-succinimida química (NHS). </li><li> Uma solução de meio livre de biotina foi feita por incubação de 20 ml de meio de cultura Vec Technologies celular (incluindo soro) com 200 ul de contas de streptavadin de 12 hr. As esferas foram removidas do meio de um íman e o meio foi filtrado através de um filtro de 0,22 ^ M estéril. </li><li> A câmara de fluxo foi removido da placa de cultura de células e as células foram lavadas em meio de 37 ° C a biotina livre. </li><li> Uma solução de reserva de 1 ul de 2,4 uM esferas revestidas com estreptavidina, em 1 ml de meio isento de biotina foi preparada, e 100 uL do stock foi adicionada à placa de cultura de células. </li><li> Contas de estreptavidina foram colhidos com o cantilever, enviando a ponta sobre a superfície do vidro ao lado de uma retracção, grânulo, o posicionamento da ponta do cantilever sobre o talão, e, em seguida, premir o cantilever para baixo sobre os segundos e talão de descanso para vários. </li><li> A sensibilidade do cantilever foi medidad, recuando para a região de vidro nu e usando o declive da curva para definir a deflexão da ponta como uma função da tensão. </li><li> A constante de mola do cantilever foi então calculada a partir de uma calibração térmica no software MFP3D. </li><li> O cantilever calibrado foi então utilizado para recuar as amostras, tal como mostrado na Figura 2. Estes grânulos de 2,4 uM oferecer uma maior área de contacto com a superfície da célula de modo a que as propriedades mecânicas da camada de glicocálice macio pode ser detectado. O cantilever foi colocada sobre uma célula de perto do núcleo celular e uma abordagem suave da ponta para a terceira célula foi usada para ajustar a altura do braço de suporte cerca de 3 um acima da superfície da célula. O software foi ajustado para 20 indentações repetidas a uma taxa de 1 um / seg a uma força máxima de 7 nN. Aproximadamente 6 segundos decorrido entre contactos sucessivos. É possível a utilização de diferentes taxas de recuo para testar as propriedades dependentes do tempo do glicocálice, embora nestes inicial<html> experiências, apenas uma taxa única indentação (1 um / seg) foi usada. </li></ol> 2. Teoria recuo Indentação em um meio-espaço elástico com uma esfera de raio R pode ser descrito utilizando teoria de Hertz, onde a força de recuo, F, é dada pela equação: <img alt="Equação 2" fo:content-width="1.157in" fo:src="/files/ftp_upload/50163/50163eq2highres.jpg" src="/files/ftp_upload/50163/50163eq2.jpg" /> Onde δ é a profundidade de indentação e E * é o módulo de redução do material a ensaiar (Figura 3). No caso de um penetrador infinitamente rígida que colide um uniforme elástica semi-espaço, E * é dado pela equação: 3/50163eq3.jpg &quot;/&gt; onde E é o módulo elástico e ν é o coeficiente de Poisson do material. Trabalhos recentes com películas de polímeros tem inspirado o desenvolvimento de um modelo de duas camadas para a determinação do módulo de elasticidade e espessura de filmes finos 1. Estamos a aplicar este modelo para a biologia celular, tratando o glicocálice como uma película fina e uniforme macia na superfície do corpo da célula. Usando este modelo, o módulo de elasticidade reduzido do sistema torna-se: <img alt="Equação 4" fo:content-width="2.85in" fo:src="/files/ftp_upload/50163/50163eq4highres.jpg" src="/files/ftp_upload/50163/50163eq4.jpg" /> Onde E é o módulo de GC do glicocálice, células E é o módulo de elasticidade do corpo da célula, P, Q e n são constantes que foram determinadas empiricamente a partir do polímero se encaixa, e z é dado pela equação: <p class="jove_content" fo:keep-Together.within-page = &quot;always&quot;&gt; <img alt="Equação 5" fo:content-width="1.257in" fo:src="/files/ftp_upload/50163/50163eq5highres.jpg" src="/files/ftp_upload/50163/50163eq5.jpg" /> Onde t é a espessura da camada de glicocálice. Um esquema destes parâmetros é mostrado na Figura 3. O modelo tem sido demonstrado ser um modo adequado para determinar o módulo de elasticidade e espessura de uma película fina no substrato mais rígido 1. Esta equação pode ser usada para ajustar as curvas obtidas a partir de indentação em células para determinar o módulo de elasticidade e espessura da glicocálice endotelial, como mostrado na 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>

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Foram utilizados os valores calculados a partir do modelo de duas camadas e teoria de Hertz para modelar a interacção de um dos leucócitos em circulação no sangue com a parede endotelial. Calculamos que um microvilosidades na leucócitos com um diâmetro de 50 nm sob uma carga de 10 iria pN travessão aproximadamente 150 nm para o glicocálice, apenas uma fracção da espessura total. Isto indica que o glicocálice, com propriedades como medidos nesta experiência, é uma barreira significativa para a interacção ...

<table border="1"><tbody><tr><td> Nome do reagente / Material </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Meio de McCoy </td><td> Gibco </td><td> 16600-082 </td><td></td></tr><tr><td> Fetal Calf Serum </td><td> Hyclone </td><td> SH30070 </td><td></td></tr><tr><td> Meio de Crescimento de Células Endoteliais </td><td> Vec Technologies </td><td> MCDB-131 </td><td></td></tr><tr><td> Agrupados células endoteliais da veia umbilical humana </td><td> Vec Technologies </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 Hidrogênio </td><td> VWR </td><td> BDH3742-1 </td><td></td></tr><tr><td> (3-aminopropil) trietoxissilano </td><td> Aldrich </td><td> 440.140-100ML </td><td></td></tr><tr><td> Álcool 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> Solução salina tamponada de Hank </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> Contas Streptavadin </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 Cantilever sem </td><td> Nanoworld </td><td> Arrow-TL1-50 </td><td></td></tr><tr><td> Silhouette SD </td><td> Quickutz </td><td> Silhueta-SD </td><td></td></tr><tr><td> Borracha de silicone </td><td> Stockwell Elastomerics </td><td> SE50-RS </td><td></td></tr><tr><td> Seringas 30 ml </td&gt; <td> Benton Dickinson </td><td> 309650 </td><td></td></tr><tr><td> 18 agulhas de calibre </td><td> Benton Dickinson </td><td> 305196 </td><td></td></tr><tr><td> Conjuntos de extensão </td><td> Hospira </td><td> 4429-48 </td><td></td></tr><tr><td> Válvulas de 4 vias </td><td> Teleflex </td><td> W21372 </td><td></td></tr><tr><td> Masculino / Feminino Caps Porto </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> Tubulação Peristaltic </td><td> Watson-Marlow </td><td> 903.0016.016 </td><td></td></tr><tr><td> filtros estéreis </td><td> Ciências da Vida Pall </td><td> 4652 </td><td></td></tr></tbody></table>

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

Nossa compreensão da interacção de leucócitos e da parede do vaso durante a captura de leucócitos é limitada por uma compreensão incompleta das propriedades mecânicas da camada de superfície endotelial. Sabe-se que as moléculas de adesão nos leucócitos estão distribuídos de maneira não uniforme em relação à superfície topografia 3, que a topografia limita a formação da ligação adesiva com superfícies de outros 9, e que as forças de contacto fisiológicas (≈ 5,0-10,0 pN por microvilosidades) pode comprimir os microvilos como menos de um terço do seu comprimento de repouso, o aumento da acessibilidade das moléculas para a superfície oposta 3, 7. Consideramos que o endotélio como uma estrutura de duas camadas, o corpo da célula, relativamente rígida, mais o glicocálice, um revestimento de açúcar mole protectora na superfície luminal 6. Tem sido demonstrado que o glicocálice pode actuar como uma barreira para reduzir a adesão de leucócitos ao endotélio de superfície 4.Neste relatório, nós começamos a abordar a deformabilidade de superfícies endoteliais para entender como a rigidez mecânica endotelial pode afetar a formação de vínculo. As células endoteliais em cultura estática não expressam uma glicocálice robusto, mas as células cultivadas em condições de fluxo fisiológicas começam a aproximar o glicocálice observada in vivo 2. O módulo de elasticidade do corpo de célula endotelial foi medida através de microscopia de força atómica (AFM) ser aproximadamente de 5 a 20 kPa 5. A espessura e estrutura do glicocálice foram estudadas utilizando microscopia electrónica de 8, e o módulo de elasticidade do glicocálice foi aproximada usando métodos indirectos, mas o nosso conhecimento, não existem relatos na literatura de uma medição directa do módulo glicocálice em células vivas . No presente estudo, nós apresentamos experiências feitas com um recuo AFM novel sonda em células que foram cultivadas em condições de maximizar a sua expressão glicocálice de maas medições directas ke do módulo e da espessura do glicocálice endotelial.

Numa experiência típica, 20 força-vs-distância curvas foram obtidas a partir de uma dada região de célula, tipicamente na região perinuclear, próximo, mas não sobre, o núcleo (dentro de ~ ^ M 2). As curvas foram alinhados para levar em conta qualquer variação de amostra ao longo da duração da medição e, em seguida, em média, para remover o ruído cantilever, como mostrado na Figura 4. As curvas foram analisadas e se encaixam com o modelo de duas camadas que foi desenvolvido para determi...

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

As características mecânicas do glicocálice endotelial foi medida através da indentação utilizando esferas micronizadas em cantilevers de AFM. As células endoteliais foram cultivadas numa câmara de costume em condições de fluxo fisiológicas para induzir a expressão de glicocálice. Os dados foram analisados ​​utilizando um modelo de película fina, para determinar a espessura e módulo de glicocálice.

Quantificar as propriedades mecânicas do Glicocálix endotelial com Microscopia de Força Atômica

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

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13.1K visualizações.  University of Rochester. As características mecânicas do glicocálice endotelial foi medida através da indentação utilizando esferas micronizadas em cantilevers de AFM. As células endoteliais foram cultivadas numa câmara de costume em condições de fluxo fisiológicas para induzir a expressão de glicocálice. Os dados foram analisados ​​utilizando um modelo de película fina, para determinar a espessura e módulo de glicocálice.

Vídeo: 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).

A imobilização de bactérias for Imaging por Microscopia de Força Atômica

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 ...

Bioengenharia

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.

Visualização do DNA recombinante e Complexos de Proteínas Usando Microscopia de Força Atômica

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).

Medir as propriedades mecânicas das células vivas Usando Microscopia de Força Atômica

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.

Um novo método para localizar Repórter fluorescentes Beads perto da superfície de cultivo celular para Tração Microscopia de Força

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.

Projeção de imagem atômica e força Espectroscopia de Bicamadas Lipídicas suportados

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.

Em Situ Mapeamento das propriedades mecânicas de biofilmes por de rastreamento de partículas 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.

Síntese de hidrogeles com propriedades anti-deposição como membranas de purificação de água

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.

Um milímetro escala Flexural sistema de testes para medir as propriedades mecânicas de espículas de esponja marinha

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.

Biaxial caracterizações mecânicas das válvulas cardíacas Atrioventricular

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).

Aplicação de microscopia de força atômica para detectar osteoartrite precoce

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