Gli autori desiderano ringraziare Elena Lomakina, Richard Bauserman, Margaret Youngman, Shay Vaknin, Jessica Snyder, Chris Striemer, Nakul Nataraj, Hung Li Chung, Tejas Khire, e Eric Lam per la loro assistenza in questo progetto. Questo progetto è stato finanziato dal NIH # PO1 HL 018208.

1. Metodi 1,1 Cella camera di flusso Una camera di flusso, illustrato in figura 1, è stato costruito in modo che le cellule possono essere coltivate in un taglio di 1,0 Pa (10 dyn / cm 2) e poi trasferita direttamente a un AFM Asylum MFP3D (Santa Barbara, CA). <ol><li> La camera di flusso è stato preparato per l&#39;esperimento prima pulizia dei vetrini in soluzione Piranha (3:1 H 2 SO 4: H 2 O 2) per 15 min e poi lavando con acqua distillata. Essi sono stati poi cotto a secco e rivestita con trietossi amminopropil silano (APTES) in una camera di deposizione sotto vuoto. </li><li> Una guarnizione in silicone è stato tagliato con un utensile da taglio Silhouette SD. Questo ha permesso di controllare con precisione le dimensioni della camera di flusso per il controllo della portata e sollecitazione di taglio durante la crescita cellulare. Tipicamente, un canale è stato tagliato 6,4 millimetri di larghezza per 19 mm di lunghezza da un foglio di silicone 0,4 mm. La portata necessaria per generite una sollecitazione di taglio di 1,0 Pa (10 dyn / cm 2) è calcolato assumendo flusso laminare in un canale rettangolare con l&#39;equazione: </li></ol><img alt="Equazione 1" fo:content-width="0.737in" fo:src="/files/ftp_upload/50163/50163eq1highres.jpg" src="/files/ftp_upload/50163/50163eq1.jpg" /> dove Q è la portata, τ è la tensione tangenziale, μ è la viscosità del mezzo, assume qui per essere 1,0 mPa (0,01 sec * dyn / cm 2), h è l&#39;altezza e w è la larghezza della camera di flusso . <ol start="3"><li> La parte superiore della camera di flusso è stata allineata con la guarnizione nella piastra di coltura cellulare e fissato con un anello magnetico. Il gruppo è stato riempito con alcol isopropilico (IPA) per la sterilizzazione. </li><li> Il sistema a flusso totale è stato assemblato. Le porte di flusso nella piastra di coltura cellulare sono stati collegati a tre vie. Le valvole sono state collegate ad aprire siringa da 30 mls. IPA è stato lavato attraverso il sistema, che è stato poi lavato con 30 ml di mezzo di McCoy con 4% di siero fetale bovino (FCS). Il sistema è stato riempito con 20 ml di mezzo di crescita cellulare Vec Technologies. Copertine sono stati collocati sulle cime delle siringhe. Il tappo del serbatoio cattura siringa aveva un ago in atto per spostare il supporto al serbatoio di alimentazione. 0,2 micron filtri sterili erano attaccati alle prese d&#39;aria dei coperchi per evitare la contaminazione del sistema. La camera di flusso è quindi pronto per semina cellulare. </li></ol> 1,2 Colture Cellulari <ol><li> Cellule endoteliali della vena ombelicale (HUVEC) e terreno di coltura sono stati acquistati da Technologies Vec (Rensselaer, NY) e coltivate a confluenza in una beuta T25. </li><li> Il mezzo di crescita è stato rimosso dal pallone e il monostrato di cellule rilasciate con 2 ml di 2,5% tripsina. Una volta che le cellule erano in soluzione, la tripsinizzazione è stata bloccata con l&#39;aggiunta di 10 ml di mezzo di coltura cellulare al pallone. </li><li> Thsospensione cellulare è stata centrifugata e per 5 minuti ed il surnatante è stato rimosso. Le cellule sono state risospese in 1 ml di mezzo di coltura cellulare (contenente siero) per l&#39;iniezione nella camera di flusso. </li><li> La sospensione cellulare (0,5 ml, ~ 50.000 cellule) è stato caricato in una siringa e iniettato nella camera di flusso attraverso una valvola a tre vie. </li><li> Le cellule sono state lasciate sedimentare e aderire al substrato di vetro per 2 h prima di flusso è stato avviato. Le cellule sono state cresciute in condizioni di flusso in un incubatore a 37 ° C per 1 a 5 giorni fino confluenti. </li></ol> 1.3 Preparazione a sbalzo e rientro cellulare <ol><li> Tipless cantilever AFM (nanomondo, Svizzera) sono stati puliti in acido nitrico per 5 min e funzionalizzato con amminopropil trietossi silano (APTES) in una camera di deposizione vapore. </li><li> Una soluzione di 5 mg / ml in peso NHS-solfo-LC-biotina in soluzione salina tamponata di Hank (HBSS) è stato preparato. I cantilever sono stati immersi nella soluzione per 15 minuti per coniugatocancello il silano con N-Hydroxysuccinimide (NHS) chimica. </li><li> Una soluzione di mezzo di biotina libera è stata effettuata incubando 20 ml di mezzo di coltura cellulare Vec Technologies (compreso il siero) con 200 microlitri di perline streptavadin per 12 hr. Le perle sono state rimosse dal mezzo di magnete e il mezzo è stato filtrato attraverso un filtro di 0,22 micron sterile. </li><li> La camera di flusso è stato rimosso dalla piastra di coltura cellulare e le cellule sono state lavate in 37 ° C biotina medie. </li><li> Una soluzione stock di 1 ml di 2,4 micron perle rivestite con streptavidina in 1 ml di mezzo di biotina libera è stato preparato, e 100 pl di magazzino sono stati aggiunti alla piastra di coltura cellulare. </li><li> Perline streptavidina stati prelevati con il cantilever di atterraggio della punta sulla superficie del vetro accanto ad un tallone, ritrarre, posizionando l&#39;apice del cantilever sul tallone, e quindi premendo il cantilever giù sulle secondo tallone e di riposo per diversi. </li><li> La sensibilità della misura era cantileverd indentando su una regione di vetro nuda e utilizzando la pendenza della curva di impostare la deflessione punta in funzione della tensione. </li><li> La costante elastica del cantilever stato quindi calcolato da una calibrazione termica nel software MFP3D. </li><li> Il cantilever calibrato è stato poi utilizzato per rientro dei campioni, come mostrato in Figura 2. Queste perle 2,4 micron offrono una maggiore superficie di contatto con la superficie cellulare in modo che le proprietà meccaniche dello strato morbido glicocalice può essere rilevato. Il cantilever è stato posizionato sopra una cella vicino al nucleo cellulare e un approccio morbido del puntale nella cella è stato usato per impostare l&#39;altezza a sbalzo circa 3 micron sopra la superficie cellulare. Il software è stato impostato per 20 rientranze ripetute ad una velocità di 1 um / sec ad una forza massima di 7 nN. Circa 6 secondi trascorsi tra i contatti successivi. È possibile utilizzare diversi tassi di rientro per verificare dipendenti dal tempo proprietà del glicocalice, anche se in questi primi<html> esperimenti, solo un tasso rientranza singola (1 um / sec) è stato usato. </li></ol> 2. Teoria indentazione Rientro in un semispazio elastico con una sfera di raggio R possono essere descritti usando teoria di Hertz in cui viene data la forza di rientro, F, mediante l&#39;equazione: <img alt="Equazione 2" fo:content-width="1.157in" fo:src="/files/ftp_upload/50163/50163eq2highres.jpg" src="/files/ftp_upload/50163/50163eq2.jpg" /> Dove δ è la profondità di penetrazione e * E è il modulo ridotto del materiale in prova (Figura 3). Nel caso di un penetratore infinitamente rigida interferire uniforme elastica semispazio, E * è data dall&#39;equazione: 3/50163eq3.jpg &quot;/&gt; dove E è il modulo elastico e ν è il rapporto di Poisson del materiale. Recente lavoro con polimero film ha ispirato lo sviluppo di un modello a due strati per la determinazione del modulo e lo spessore di film sottili 1. Stiamo applicando questo modello alla biologia cellulare trattando il glicocalice come una pellicola sottile uniforme morbido sulla superficie del corpo cellulare. Utilizzando questo modello, il modulo ridotta del sistema diventa: <img alt="Equazione 4" fo:content-width="2.85in" fo:src="/files/ftp_upload/50163/50163eq4highres.jpg" src="/files/ftp_upload/50163/50163eq4.jpg" /> Dove E è il modulo GC del glicocalice, cella E è il modulo del corpo cellulare, P, Q ed n sono costanti che sono stati determinati empiricamente dal polimero adatto, e Z è data dall&#39;equazione: <p class="jove_content" fo:keep-Together.within-page = &quot;always&quot;&gt; <img alt="Equazione 5" fo:content-width="1.257in" fo:src="/files/ftp_upload/50163/50163eq5highres.jpg" src="/files/ftp_upload/50163/50163eq5.jpg" /> Dove t è lo spessore dello strato di glicocalice. Uno schema di questi parametri è mostrato in Figura 3. Il modello è stato dimostrato di essere un modo accurato per determinare il modulo e lo spessore di una pellicola sottile su substrato rigido 1. Questa equazione può essere utilizzata per adattare le curve ottenute dal rientro in cellule per determinare il modulo e lo spessore del glicocalice endoteliale, come mostrato in 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>

Nessun conflitto di interessi dichiarati.

Abbiamo usato valori calcolati dal modello a due strati e teoria Hertz per modellare l&#39;interazione di un leucociti circolanti nel sangue con la parete endoteliale. Abbiamo calcolato che un microvillus sulla leucociti con un diametro di 50 nm sotto un carico di 10 pN sarebbe trattino circa 150 nm nel glicocalice, solo una frazione dello spessore totale. Ciò indica che il glicocalice, con proprietà misurate in questo esperimento, è una barriera significativa interazione cellula-cellula e può essere un grande ostac...

<table border="1"><tbody><tr><td> Nome del reagente / materiale </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Mezzo di 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> Cellule endoteliali terreno di crescita </td><td> Vec Technologies </td><td> MCDB-131 </td><td></td></tr><tr><td> Pool umani ombelicale cellule endoteliali Vena </td><td> Vec Technologies </td><td> PHUVEC/T-25 </td><td></td></tr><tr><td> Acido solforico </td><td> JT Baker </td><td> 9681-02 </td><td></td></tr><tr><td> Perossido di idrogeno </td><td> VWR </td><td> BDH3742-1 </td><td></td></tr><tr><td> (3-amminopropil) triethoxysilane </td><td> Aldrich </td><td> 440140-100ML </td><td></td></tr><tr><td> Alcool isopropilico </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> Sale di Hank soluzione tamponata </td><td> Gibco </td><td> 14175-095 </td><td></td></tr><tr><td> solfo-NHS-LC-biotina </td><td> Thermo Scientific </td><td> 21335 </td><td></td></tr><tr><td> Streptavadin perline </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> Mensole Tipless </td><td> Nanomondo </td><td> ARROW-TL1-50 </td><td></td></tr><tr><td> Silhouette SD </td><td> Quickutz </td><td> Silhouette-SD </td><td></td></tr><tr><td> Gomma di silicone </td><td> Stockwell elastomeri </td><td> SE50-RS </td><td></td></tr><tr><td> 30 ml Siringhe </td&gt; <td> Benton Dickinson </td><td> 309650 </td><td></td></tr><tr><td> Calibro 18 aghi </td><td> Benton Dickinson </td><td> 305196 </td><td></td></tr><tr><td> Set di estensione </td><td> Hospira </td><td> 4429-48 </td><td></td></tr><tr><td> 4 vie </td><td> Teleflex </td><td> W21372 </td><td></td></tr><tr><td> Maschio / femmina Port Caps </td><td> Smith Medical </td><td> MX491B </td><td></td></tr><tr><td> Pompa peristaltica </td><td> Watson-Marlow </td><td> 401U / D </td><td></td></tr><tr><td> Tubi peristaltica </td><td> Watson-Marlow </td><td> 903.0016.016 </td><td></td></tr><tr><td> filtri sterili </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

Nostra comprensione dell&#39;interazione dei leucociti e la parete del serbatoio durante la cattura dei leucociti è limitata da una non completa comprensione delle proprietà meccaniche dello strato di superficie endoteliale. È noto che molecole di adesione sui leucociti sono distribuite in modo non uniforme rispetto alla superficie topografia 3, che limita la formazione topografia adesivo legame con altre superfici 9, e che le forze di contatto fisiologici (≈ 5,0-10,0 pN per microvillus) può comprimere i microvilli come meno un terzo della loro lunghezza a riposo, aumentando l&#39;accessibilità di molecole alla superficie opposta 3, 7. Consideriamo l&#39;endotelio come struttura a due strati, il corpo cellulare relativamente rigido, più il glicocalice, un rivestimento morbido zucchero protettivo sulla superficie luminale 6. E &#39;stato dimostrato che il glicocalice può agire come una barriera per ridurre l&#39;adesione dei leucociti al 4 superficie endoteliale.In questa relazione si comincia ad affrontare la deformabilità delle superfici endoteliali per capire come la rigidità meccanica endoteliale potrebbe influenzare la formazione del legame. Le cellule endoteliali in coltura statica non esprimono un glicocalice robusta, ma le cellule cresciute in condizioni di flusso fisiologiche cominciano ad approssimare il glicocalice osservate in vivo 2. Il modulo del corpo cellulare endoteliale è stata misurata utilizzando la microscopia a forza atomica (AFM) da circa 5 a 20 kPa 5. Lo spessore e la struttura del glicocalice sono stati studiati mediante microscopia elettronica 8, e il modulo del glicocalice è stato calcolato utilizzando metodi indiretti, ma a nostra conoscenza, non ci sono stati rapporti pubblicati di una misura diretta del modulo glicocalice in cellule viventi . In questo studio, presentiamo indentazione esperimenti effettuati con una nuova sonda AFM sulle cellule che sono state coltivate in condizioni di massimizzare la loro espressione di glicocalice make misurazioni dirette del modulo e lo spessore del glicocalice endoteliale.

In un esperimento tipico, 20 forza-vs-distanza curve sono state ottenute da una data regione della cellula, tipicamente nella regione perinucleare, vicino, ma non, il nucleo (entro ~ 2 micron). Le curve sono state allineate per tenere conto di qualsiasi deriva campione per tutta la durata della misurazione e poi mediati per rimuovere il rumore cantilever, come mostrato in Figura 4. Le curve sono state analizzate e forma con il modello a due strati che è stato sviluppato per la determinazione del modulo...

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

Le caratteristiche meccaniche del glicocalice endoteliale sono state misurate utilizzando sfere indentazione micron di dimensioni su cantilever AFM. Le cellule endoteliali sono state coltivate in una camera personalizzata in condizioni fisiologiche di flusso per indurre l&#39;espressione glicocalice. I dati sono stati analizzati utilizzando un modello di pellicola sottile per determinare lo spessore glicocalice e modulo.

Quantificare le proprietà meccaniche del glicocalice endoteliale con microscopia a forza atomica

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 Views.  University of Rochester. Le caratteristiche meccaniche del glicocalice endoteliale sono state misurate utilizzando sfere indentazione micron di dimensioni su cantilever AFM. Le cellule endoteliali sono state coltivate in una camera personalizzata in condizioni fisiologiche di flusso per indurre l'espressione glicocalice. I dati sono stati analizzati utilizzando un modello di pellicola sottile per determinare lo spessore glicocalice e modulo.

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).

Immobilizzazione batterica per Imaging di microscopia a forza atomica

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 ...

Ricerca

Bioingegneria

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.

Visualizzazione del DNA ricombinante e Complessi proteina usando la microscopia a forza atomica

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).

Misurare le proprietà meccaniche del Living cellule utilizzando microscopia a forza atomica

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 nuovo metodo per la localizzazione Reporter perline fluorescenti vicino alla superficie coltura cellulare per la trazione microscopia a forza

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.

Atomic Force Microscopy Imaging e Spettroscopia Forza supportati doppi strati lipidici

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.

In Situ mappatura delle proprietà meccaniche del biofilm da-Particle tracciamento 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.

Sintesi di idrogel con proprietà antivegetative come membrane per la purificazione dell&#39;acqua

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 sistema di prova millimetro scala flessionale per misurare le proprietà meccaniche di spicole di spugna 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.

Caratterizzazioni biassiale meccaniche delle valvole atrioventricolari cuore

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).

Applicazione della microscopia a forza atomica per rilevare l'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 ...