This work was partly supported by NSF CBET 1227766, NSF CBET 1511759, and Byars-Tarnay Endowment. We gratefully acknowledge use of the West Virginia University Shared Research Facilities which are supported, in part, by NSF EPS-1003907.

1. Die Replikation von PDMS-Formen aus einer EBL Mold <ol><li> Fabrizieren Silikonform 29 <ol><li> Schleuderbeschichtungs 200 ul Polymethylmethacrylat (PMMA) -Lösung auf einem 1 × 1 cm Silizium (Si) -Substrat bei 2.500 rpm für 1 min um einen dünnen Film zu bilden. </li><li> Backen die PMMA-Film auf dem Si-Substrat bei 180 ° C für 2 min. </li><li> Schreiben die entworfen Nano auf der PMMA - Film durch einen fokussierten Elektronenstrahl bei einer Flächendosis von 300 &amp; mgr; C / cm 2. </li><li> Entwickeln Sie die PMMA-Nano in Entwickler für 80 Sekunden. </li><li> Hinterlegung der PMMA Nano mit einer Nickelschicht von 50 nm Dicke in eine E-beam Verdampfer bei einer Ausgangsspannung von 10 kV, Emissionsstrom von 0,5 mA und einer Abscheidungsrate von 0,5 Å / sec Verwendung. </li><li> Heben Sie den PMMA Teil in 20 ml-Entferner bei 80 ° C für 20 min ab. </li><li> Reaktives Ionenätzen (RIE) der Nano in das Si-Substrat eine Si Form der gewünschten Tiefe zu erhalten. HINWEIS: Gasgemisch aus TetRafluoromethane (CF 4) / Sauerstoff (O 2) (90% / 10%) bei einem induktiv gekoppelten Plasma (ICP) Leistung von 400 W und RIE Leistung von 150 W verwendet wird Si - Substrat zu einer Tiefe von 560 nm zu ätzen. </li></ol></li><li> Silanisieren Si Form <ol><li> Setzen Sie ein Deckglas und die Si-Form in einem 100 mm PS Petrischale und übertragen sie in einem Glas-Exsikkator in einer Abzugshaube. </li><li> Drop 10 ul 1H, 1H, 2H, 2H-Perfluorooctyltrichlorosilane auf dem Deckglas. Achtung: 1H, 1H, 2H, 2H-Perfluorooctyltrichlorosilane verursachen Haut Korrosion und schwere Augenschäden. Tragen Sie geeignete Schutzausrüstung (PSA). </li><li> Decken Sie die Petrischale teilweise. </li><li> Halten Sie den Exsikkator unter Vakuum für 5 Stunden in einem Abzug der Silanisierung der Si Form zu vervollständigen. </li></ol></li><li> Bereiten Sie PDMS-Präpolymer <ol><li> Man wiegt 10 g PDMS-Harz und 1,05 g Härter in einem Einweg-Wägeschiffchen. </li><li> Mischen Sie die PDMS-Präpolymer gründlich durch einen Plastiklöffel verwenden. </li><li> Entgasen Sie den PDMS-Präpolymer in einem Kunststoff-Exsikkator unter Vakuum etwa 20 min bis eine klare Mischung beobachtet wird. </li></ol></li><li> Replizieren PDMS Formen <ol><li> Setzen Sie die silanisierten Si Form in einer 60 mm Petrischale. </li><li> Gießen Sie die PDMS-Präpolymer auf der Si-Form in der Petrischale. </li><li> Legen Sie die Petrischale in einem Kunststoff-Exsikkator und Entgasen für etwa 10 Minuten, bis alle Blasen verschwinden. </li><li> Übertragen Sie die Petrischale auf einer Heizplatte und Härten des PDMS-Präpolymer bei 70 ° C für 4 Stunden. </li><li> Ziehen Sie die PDMS-Form von der Si-Form aus sorgfältig von einer Pinzette. HINWEIS: PDMS Formen können für bis zu einer Woche bei Umgebungsbedingungen gelagert werden. Nach dem Aushärten gibt es einige nicht vernetzten PDMS Harzmoleküle und restlichem Vernetzungsmittel in den PDMS Formen 30. Die niedermolekularen Molekülen allmählich diffundieren und sammeln sich an der Oberfläche über die Zeit. Dies wirkt sich auf die topographischen und mechanischen Eigenschaften der PDMS - Oberfläche 31. Die difFusion ist nicht signifikant innerhalb einer Woche. </li></ol></li></ol> 2. Nähen von PDMS-Formen in einem großen, einzigen Form <ol><li> Bereiten Sie mehrere PDMS Formen für Schritt 1.4 wiederholen. HINWEIS: Wiegen gleiche Menge an PDMS-Mischung jedesmal PDMS Formen von gleicher Dicke zu erhalten. </li><li> Bestimmen Sie die Ausrichtung der anisotropen PDMS Nano wie nanogratings unter einem optischen Mikroskop und markieren Sie ihn auf der Rückseite der PDMS-Formen mit einem Filzstift. HINWEIS: Es ist nicht notwendig, die Orientierung der isotropen Nanotopographie, wie beispielsweise Nanosäulen zu markieren. </li><li> Reinigen Sie ein Si-Substrat mit Ethanol in einer Abzugshaube und trocknen Sie sie mit Druckluft. </li><li> Schneiden Sie die nicht gemusterten Flächen jedes PDMS-Form mit einem Messer. HINWEIS: Für die PDMS Formen, die an der Peripherie der genähten Form gelegt wird, werden nur die ungemusterten Bereiche in Kontakt mit anderen abgeschnitten werden sollten. </li><li> Legen Sie die getrimmten PDMS-Form mit dem Nano Gesicht nach unten aufdie Spiegelseite des Si-Substrats und dann auszurichten andere Formen der Nähe von, aber nicht die umgebende Form (en) zu berühren. </li><li> Bereiten Sie eine PDMS Klebeschicht <ol><li> PDMS-Präpolymer (PDMS Harz und Härter-Verhältnis: 10: 1,05) gegossen 1 g entgastes auf einem sauberen Objektträger aus Glas (7,5 cm × 2,5 cm), um eine 0,5 mm dicke Schicht zu bilden. </li><li> Backen Sie die PDMS-Schicht bei 100 ° C auf einer Heizplatte für 3-5 min. Verwenden Sie eine Nadel, um die Schicht zu berühren und zu gewährleisten, dass die Schicht teilweise, aber nicht vollständig geheilt. HINWEIS: teilweise gehärtete PDMS kann nicht wie nicht gehärtete PDMS-Präpolymer fließen, aber es ist klebrig, verglichen mit geheilt PDMS. </li></ol></li><li> Legen Sie die PDMS-Schicht auf der Rückseite ausgerichtet PDMS Formen und schnell diese Baugruppe invertieren und übertragen sie auf der Heizplatte. </li><li> Übernehmen einer Druckkraft (5 kPa) unter Verwendung eines Metallblock auf der Oberseite des Montage einen guten Kontakt zwischen dem PDMS Haftschicht und der Rückseite des PDMS Formen zu gewährleisten, und härten die PDMS Klebstoffschicht bei 100 ° C für 1 Std. &lt;br /&gt; Hinweis: Sie sorgfältig die Position des Metallblocks stellen Sie die Neigung der Anordnung zu vermeiden. </li><li> Entfernen Sie den Metallblock und ziehen Sie die einzelnen, genäht PDMS-Form aus dem Si-Substrat ab. </li></ol> 3. Erzeugung eines Master-Form auf PS Substrate Hinweis: Die genähte PDMS-Form auf einem Glasträger immobilisiert kann verwendet werden, eine Mutterform auf einer PS-Platte oder eine PS-Dünnfilm zu erzeugen, aus dem Arbeitsnanostrukturierten Substrate hergestellt werden können. <ol><li> Generieren Sie eine Masterform auf einer PS-Platte <ol><li> Bereiten Sie eine PS-Platte <ol><li> Trocknen Sie die PS-Pellets in einem Vakuumofen bei 80 ° C für zwei Tage. </li><li> Vorheizen eine Pressmaschine bei 230 ° C. </li><li> Bauen Sie eine Aluminiumplatte, eine Polytetrafluorethylen (PTFE) Blatt und das Aluminium-Abstandhalter in einer Reihenfolge von unten nach oben. </li><li> Belastung 3,5 g PS-Pellets in der Aluminiumabstandshalter mit einer quadratischen Öffnung von 3 cm (L) · 3 cm (W) × 0,3 cm (H). HINWEIS: Der Abstandhalter ist etwa 0.1 cm dicker als die PDMS Formen, und somit die endgültige nanostrukturierten PS Substrat beträgt etwa 0,1 cm dick. </li><li> Legen Sie eine andere PTFE-Folie und dann eine andere Aluminiumplatte auf dem Aluminium-Abstandhalter. </li><li> Legen Sie die Montage in der Pressmaschine. </li><li> Vorwärmen der PS-Pellets bei 230ºC für 30 min. </li><li> Tragen Sie einen Kompressionsdruck (0,1 MPa) auf der Baugruppe für 5 min. </li><li> Lassen Sie den Druck und dann einen Kompressionsdruck von 0,5 MPa auf die Montage erneut anwenden. </li><li> Wiederholen Sie Schritt 3.1.1.9 mit einem Druckanstieg von 0,5 MPa bis der erwünschte Druck von 1,5 MPa erreicht ist. </li><li> Schalten Sie die Heizung der Pressmaschine ab und kühlen es bei einem konstanten Druck von 1,5 MPa unter 70 ° C nach unten. </li><li> Nehmen Sie die Baugruppe aus und speichern Sie die PS-Platte in einem Vakuumofen bei 80 ° C Feuchtigkeit zu verhindern, dass der PS-Platte erneut eingeben. </li></ol></li><li> Nanoimprint die genähte PDMS-Form in PS-Platte <ol><li> Legen Sie die PS-Platte in einem Aluminium-AbstandhalterAuf einem 3-Zoll-Si-Wafer. HINWEIS: Die Innenabmessungen des Abstandhalters sind die gleichen wie diejenigen der PS-Platte, so dass die PS-Platte passt genau in den Abstandshalter. </li><li> Erhitzen Sie die PS-Platte auf einer Heizplatte bei 250 ° C für 30 min. </li><li> Legen Sie die genäht PDMS-Form mit Nano Gesicht nach unten auf dem geschmolzenen PS-Platte. HINWEIS: Eine Seite der PDMS-Form wird in Kontakt mit der Oberfläche der PS-Platte gelegt erste und eine andere Seite abgesenkt wird allmählich in Kontakt mit der PS Oberfläche die Bildung von Luftblasen an der Grenzfläche zu vermeiden. Achtung: Die Oberfläche der Heizplatte ist heiß. Tragen Sie thermogloves während Nanoimprint Prozess. </li><li> Legen Sie eine Aluminiumplatte auf dem Glasträger der genähten PDMS-Form. </li><li> Tragen Sie einen Kompressionsdruck (12,5 kPa) durch Metallblöcke auf der Aluminiumplatte verwendet und für 3 Minuten warten. HINWEIS: Stellen Sie sicher, dass die Aluminiumplatte nicht gekippt wird. </li><li> Heben und den Metallblock von der Aluminiumplatte ersetzen und increase den Kompressionsdruck auf 25 kPa. </li><li> Wiederholen Sie Schritt 3.1.2.6 mit dem Druck stieg auf 50 kPa. HINWEIS: Dieser Schritt ist die Luft zwischen der PDMS-Form und PS-Platte gefangen zu entfernen. </li><li> Aufrechterhaltung der Temperatur der Heizplatte zwischen 240 und 250 ° C unter ständiger Druck von 50 kPa für 15 min. </li><li> Schalten Sie die Kochstelle aus und abkühlen die gesamte Einrichtung. HINWEIS: Ein Lüfter kann dazu verwendet werden, um den Kühlprozess zu beschleunigen. </li><li> Entfernen Sie die Metallblöcke, nachdem die Temperatur unter 50 ° C liegt, und schälen sorgfältig die genähte PDMS-Form aus der PS-Platte. HINWEIS: Die PS-Substrat hat die Reverse-Nano und kann als Master-Form verwendet werden, arbeiten PDMS-Substrate herzustellen. </li></ol></li></ol></li><li> Generieren Sie eine Masterform auf einem PS-Dünnfilm <ol><li> Bereiten Sie eine PS-Dünnschicht <ol><li> Man löst 1 g PS in 10 ml Toluol in einem Abzug. Vorsicht: Toluol kann Haut irritatio verursachenn und schwere Augenschäden und kann die Organe schädigen bei längerer oder wiederholter Exposition. Tragen Sie geeignete PSA. </li><li> Spin-coat 1 ml PS-Lösung auf einem 2-in-Wafer bei 2500 Upm für 1 min ~ 1 &amp; mgr; m dicken PS-Dünnfilm zu bilden. </li><li> Verdunsten Toluol aus dem Film durch die PS-Film auf Si-Wafer in einer Abzugshaube Einstellung für 3 Tage. </li><li> Einbrennlackieren PS-Dünnfilm in einem Vakuumofen über Nacht bei 80 ° C. </li></ol></li><li> Nanoimprint die PDMS-Form auf einem PS-Dünnfilm <ol><li> Legen Sie die genäht PDMS-Form mit Nanotopographie der Vorderseite nach unten auf der PS-Dünnfilm, der auf einer Heizplatte eingestellt ist. </li><li> Tragen Sie einen Kompressionsdruck von 12 kPa auf der PDMS-Form von Metallblöcke auf der Glasseite der PDMS-Form verwendet wird. </li><li> Erhöhen Sie die Temperatur der Heizplatte auf 180 ° C und halten es für 15 min. Achtung: Das geschmolzene PS Film kann als Schmiermittel wirken. Achten Sie darauf, die Metallblöcke ein Abrutschen zu verhindern. </li><li> Schalten Sie die Kochplatteund kühlen das gesamte Setup nach unten. HINWEIS: Ein Lüfter kann dazu verwendet werden, um den Kühlprozess zu beschleunigen. </li><li> Entfernen Sie die Metallblöcke, nachdem die Temperatur unter 50 ° C sinkt, und schälen sorgfältig die genäht PDMS-Form aus dem PS-Film. HINWEIS: Die nano PS Film wird als Masterform dienen Arbeits PDMS-Substrate herzustellen. </li></ol></li></ol></li></ol> 4. Nanotopographical Modulation des Zellverhaltens Hinweis: Human-Epithelzellen kultiviert werden auf die repräsentativen Nanotopographien nanotopographical Modulation von Zellausbreitung zu demonstrieren. <ol><li> Cast PDMS Arbeits Substrate aus der Masterform erzeugt entweder von Schritt 3.1 oder 3.2, je nach Anwendung. </li><li> Mit Hilfe eines hohlen Stahlbogenschlag, schneiden Sie die nanostrukturierten PDMS - Substrate in Scheiben , die die Konfiguration einer bestimmten Multi-Well - Platte (zB 24-Well - Platte) zu passen. </li><li> Verwenden einer Pinzette die PDMS-Discs in die Vertiefungen zu platzieren ofa Multi-Well-Platte. </li><li> Sterilisieren der PDMS-Substrate durch 70% Ethanol und dann UV-Exposition, die jeweils für 30 min. </li><li> Waschen Sie die PDMS-Substrate mit 1x steriler phosphatgepufferter Salzlösung (PBS) dreimal. </li><li> Beschichtung der Substrate PDMS mit extrazellulären Matrixproteins (dh 20 ug / ml Fibronektin) für 30 min bei Raumtemperatur. </li><li> Spülen Sie die PDMS-Substrate dreimal mit sterilem PBS für jeweils 5 min. </li><li> Suspend menschlichen Lungenkrebszelle A549 in Dulbeccos modifiziertem Eagle-Medium mit 10% fötalem Rinderserum und die Zellen unter Verwendung eines Hämozytometers zählen. </li><li> Platte die Zellen bei einer Aussaatdichte von 2.000 Zellen / cm 2 auf den PDMS Substrate und Kultur sie bei 37 ° C in einer befeuchteten Atmosphäre mit 5% CO 2 für einen Tag. </li><li> Waschen der Zellen mit PBS dreimal. </li><li> Fixieren Sie die Zellen in einer Mischung aus 4% Paraformaldehyd und 2% Glutaraldehyd in PBS für 4 Stunden und entwässern Zellen eine CO 2 kritischen po Verwendungint Trockner für das Scannen von elektronischen mikroskopischen Beobachtung 29. Achtung: Paraformaldehyd und Glutaraldehyd kann zu schweren Verätzungen der Haut und Augenschäden. Betreiben Sie in einer chemischen Haube und geeignete PSA tragen. </li></ol>

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Sci. 164, 111-117 (2000).</li><li>Nagase, T., Gamo, K., Kubota, T., Mashiko, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+fabrication+of+nano-gap+electrodes+by+focused+ion+beam+etching.">Direct fabrication of nano-gap electrodes by focused ion beam etching.</a> Thin Solid Films. 499 (1-2), 279-284 (2006).</li><li>Juodkazis, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+lithography+of+nanorods+in+SU-8+photoresist.">Two-photon lithography of nanorods in SU-8 photoresist.</a> Nanotechnology. 16 (6), 846 (2005).</li><li>Yang, Y., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+surfacing+for+regenerative+medicine.">Nanoscale surfacing for regenerative medicine.</a> Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2 (5), 478-495 (2010).</li><li>Yang, Y., Kulangara, K., Sia, J., Wang, L., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+a+Microfluidic+Cell+Culture+Platform+Embedded+with+Nanoscale+Features.">Engineering of a Microfluidic Cell Culture Platform Embedded with Nanoscale Features.</a> Lab Chip. 11 (9), 1638-1646 (2011).</li><li>Wang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotopographical+modulation+of+cell+function+through+nuclear+deformation.">Nanotopographical modulation of cell function through nuclear deformation.</a> Acs Appl. Mater. Inter. 8 (8), 5082-5092 (2016).</li><li>Lee, J. N., Park, C., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solvent+Compatibility+of+Poly(dimethylsiloxane)-Based+Microfluidic+Devices.">Solvent Compatibility of Poly(dimethylsiloxane)-Based Microfluidic Devices.</a> Anal. Chem. 75 (23), 6544-6554 (2003).</li><li>Yang, Y., Kulangara, K., Lam, R. T. S., Dharmawan, R., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Topographical+and+Mechanical+Property+Alterations+Induced+by+Oxygen+Plasma+Modification+on+Stem+Cell+Behavior.">Effects of Topographical and Mechanical Property Alterations Induced by Oxygen Plasma Modification on Stem Cell Behavior.</a> ACS Nano. 6 (10), 8591-8598 (2012).</li><li>Hua, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+Imprint+Lithography+with+Molecular-Scale+Resolution.">Polymer Imprint Lithography with Molecular-Scale Resolution.</a> Nano Lett. 4 (12), 2467-2471 (2004).</li><li>Delamarche, E., Schmid, H., Michel, B., Biebuyck, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+molded+polydimethylsiloxane+microstructures.">Stability of molded polydimethylsiloxane microstructures.</a> Adv. Mater. 9 (9), 741-746 (1997).</li></ol>

The authors have nothing to disclose.

Wir präsentieren Ihnen eine einfache, kostengünstige und dennoch vielseitige Methode einen großen Bereich von nanostrukturierten Substrat zu erzeugen. Um treu hochdefinierte Nano, große Aufmerksamkeit erweitern sollte auf mehrere kritische Schritte bezahlt werden. Die erste ist die mehrere PDMS Formen zu trimmen. Ungemusterten Bereiche der PDMS Formen entfernt werden müssen. Zusätzlich sollten die Seitenwände der Formen geschnitten werden vertikal so perfekt wie möglich, die Lücken zwischen den Formen zu minimi...

A number of recent findings reveal that substrate nanotopography has pronounced influence on cell behavior, from cell adhesion, spreading and migration, to proliferation and differentiation1-6. For instance, a smaller cell size and lower proliferation rate have been observed in cells cultured on deep nanogratings, even leading to apoptosis although the cell alignment, elongation and migration were enhanced, compared with the flat controls2,7-10. Moreover, nanotopography has been shown to facilitate the differentiation of stem cells into certain lineages such as neuron2,11,12, muscle13, and bone3,4. In addition, because of increasing concerns on the toxicity of engineered nanomaterials14,15, there is a need to incorporate nanotopography into physiologically relevant in vitro models for accurate risk assessment of nanomaterials. To fulfil the biochemical and molecular biology analyses, enough cells are needed to be grown on a large area of nanopatterned substrate. However, conventional nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area.
Self-assembly including colloid lithography16 and polymer demixing17 can readily generate large-area nanostructures at low costs. Because self-assembly relies on interactions between the assembling elements such as colloidal particles and macromolecules, and possible interactions between these elements and substrate, it cannot be a stand-alone method of producing nanostructures with precise spatial positioning and arbitrary shapes18. The accompanied high density of defects is also a drawback. Precise spatial control of nanopatterns can be achieved by employing templated self-assembly, which uses top-down lithographic approaches to provide the topographical and/or chemical template to guide the bottom-up assembly of the assembling elements19-21. Alternative nanofabrication techniques such as step-and-flash lithography22 and a roll-to-roll nanoimprinting lithography23 have been developed but have limited use because of their sophisticated process or the requirement of specialized equipment. Nevertheless, a template or a master mold with defined nanoscale patterns is needed for templated or alternative nanofabrication techniques. 
Such templates and master molds are conventionally generated by using focused electron, ion, or photon beam lithography. For instance, electron beam lithography (EBL)24 and focused ion beam lithography25 can generate defined patterns with a sub-5 nm resolution. Two-photon lithography has demonstrated a feature size as small as 30 nm26. Although the focused beam lithography techniques enable generation of well-defined nanoscale structures, the capital investment and the time-consuming, costly process restrict their widespread use in academic research27. Therefore, it is highly desirable to develop enabling yet affordable techniques to produce a large area of nanopatterned surfaces with high fidelity.
We have reported a simple, cost-effective stitch technique for generating a large area of nanopatterned surface from a small well-defined mold28. This protocol provides step-by-step procedure from replication of poly(dimethylsiloxane) (PDMS) molds using an EBL-written pattern, to assembly of multiple PDMS molds into a single large mold, to generation of a master mold on polymeric such as polystyrene (PS) substrates, to production of working substrates. With the expanded nanopatterned substrates, we demonstrated nanotopographical modulation of cell spreading.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>JEOL field emission SEM</td>
			<td>JEOL</td>
			<td>JSM-7600F</td>
			<td>EBL</td>
		</tr>
		<tr>
			<td>E-beam evaporator</td>
			<td>Kurt J. Lesker</td>
			<td>Model: LAB 18 e-beam evaporator</td>
			<td>nickel deposition</td>
		</tr>
		<tr>
			<td>Trion Minilock III ICP/RIE</td>
			<td>Trion technology</td>
			<td>Model: Minilock-phantom III</td>
			<td></td>
		</tr>
		<tr>
			<td>Press machine</td>
			<td>PHI Hydraulic Press</td>
			<td>Molde: SQ-230H</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>Laurell Technologies</td>
			<td>Modle: WS-400A-6NPP-LITE</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 critical dryer</td>
			<td>Tousimis</td>
			<td>Modle: Autosamdri-815</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer</td>
			<td>University Wafer</td>
			<td>1080</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum plates</td>
			<td>McMaster-carr</td>
			<td>9057K123</td>
			<td></td>
		</tr>
		<tr>
			<td>Teflon sheets</td>
			<td>McMaster-carr</td>
			<td>8711K92</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm petri dish</td>
			<td>FALCON</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm petri dish</td>
			<td>FALCON</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslip</td>
			<td>Fisher Scientific</td>
			<td>12-542-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>Fisher Scientific</td>
			<td>12-550-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable weighing boats</td>
			<td>Fisher Scientific</td>
			<td>13-735-743</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass desiccator</td>
			<td>Fisher Scientific</td>
			<td>02-913-360</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic desiccator</td>
			<td>Bel-Art Products</td>
			<td>F42025-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hotplate</td>
			<td>Fisher Scientific</td>
			<td>1110049SH</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer</td>
			<td>Ted Pella, inc.</td>
			<td>5726</td>
			<td></td>
		</tr>
		<tr>
			<td>Blade</td>
			<td>Fisher Scientific</td>
			<td>S17302</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal blocks</td>
			<td>McMaster-carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Punch</td>
			<td>Brettuns Village Leather Craft Supplies</td>
			<td>Arch punch</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(methyl methacrylate)</td>
			<td>MicroChem</td>
			<td>495 PMMA A4</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS</td>
			<td>Dow Corning</td>
			<td>Sylgard 184 kit</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene</td>
			<td>Dow Chemical</td>
			<td>Styron 685D</td>
			<td></td>
		</tr>
		<tr>
			<td>1H,1H,2H,2H-perfluorooctylmethyldichlorosilane</td>
			<td>Oakwood Chemical</td>
			<td>7142</td>
			<td></td>
		</tr>
		<tr>
			<td>Developer</td>
			<td>MicroChem</td>
			<td>MIBK/IPA at 1: 3 ratio</td>
			<td></td>
		</tr>
		<tr>
			<td>Remover</td>
			<td>MicroChem</td>
			<td>Remover PG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818500</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Fisher Scientific</td>
			<td>T324-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified eagle medium</td>
			<td>Sigma Aldrich</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11550</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microsopy Science</td>
			<td>15712-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde&#160;</td>
			<td>Fisher Chemical</td>
			<td>G151-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Corning</td>
			<td>356008</td>
			<td></td>
		</tr>
		<tr>
			<td>A549 cells</td>
			<td>ATCC</td>
			<td>ATCC CCL-185</td>
			<td></td>
		</tr>
	</tbody>
</table>

expanding nanopatterned substrates using stitch technique for nanotopographical modulation of cell behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

<html> Die Stichtechnik kann einen großen Bereich von nanostrukturierten Substrate mit hoher Genauigkeit erzeugen. Figur 1a und 1b die große Fläche von Nanostrukturen aus dem genähten PDMS - Form zu PS - Platte und PS - Dünnfilm auf einem Si - Substrat bzw. übertragen anzuzeigen. Der Vergleich zwischen dem ursprünglichen EBL geschriebene Form (Abbildung 1c) und die endgültigen PDMS Arbeitssubstrat (Abbildung 1d) b...

Watch this Scientific Journal Video about Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior at JoVE.com

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

Der Ausbau nano Substraten Stichtechnik für Nanotopographical Modulation des Zellverhaltens

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

expanding-nanopatterned-substrates-using-stitch-technique-for

Research

JoVE Journal

Bioengineering

6.5K Aufrufe.  West Virginia University. A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

Serie: Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Erstellen Zweidimensionale Patterned Substrate für Protein-und Zell-Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Forschung

Biotechnik

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Musterbildung auf Photolithographische Definierte Parylene-C: SiO<sub&gt; 2</sub&gt; Substrate

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

Ein Mikrofluidiktechnik to Cell Verformbarkeit Sonde

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (&#956;CP) is a versatile technique to pattern such grafted PEG brushes, but manual &#956;CP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-&#956;CP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal &#8216;click&#8217; chemistries. Here, we describe in detail the workflow to manufacture such substrates.

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &#181;CP and Sequential Nucleophilic Substitution

Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.

Herstellung von komplexen Kultursubstrate verwenden Robotic Mikrokontaktdrucken (R-mCP) und Sequential Nucleophile Substitution

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Ein Markierungsfreie Technik für die Raum-zeitliche Imaging von Single Cell Sekrete

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

Selektive Zellelimination aus Mixed 3D Culture Mit einem Nah-Infrarot-Technik Photoimmunotherapy

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

Messung der Steifigkeit weichem Silikon Substrate für Mechanobiology Studien mit einem Weitfeld-Fluoreszenz-Mikroskop

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Bewertung der Keratinozyten Verbreitung auf zwei- und dreidimensionale Typ I Kollagen Substrate

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Musterung der Geometrie menschlicher embryonaler Stammzellkolonien auf konformen Substraten zur Kontrolle der Gewebe-Level-Mechanik

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Formulation and Acoustic Modulation of Optically Vaporized Perfluorocarbon Nanodroplets

Microbubbles are the most commonly used imaging contrast agent in ultrasound. However, due to their size, they are limited to vascular compartments. These microbubbles can be condensed or formulated as perfluorocarbon nanodroplets (PFCnDs) that are small enough to extravasate and then be triggered acoustically at the target site. These nanoparticles can be further enhanced by including an optical absorber such as near infrared organic dye or nanoparticles (e.g., copper sulfide nanoparticles or gold nanoparticles/nanorods). Optically tagged PFCnDs can be vaporized through laser irradiation in a process known as optical droplet vaporization (ODV). This process of activation enables the use of high boiling point perfluorocarbon cores, which cannot be vaporized acoustically under the maximum mechanical index threshold for diagnostic imaging. Higher boiling point cores result in droplets that will recondense after vaporization, resulting in "blinking" PFCnDs that briefly produce contrast after vaporization before condensing back into nanodroplet form. This process can be repeated to produce contrast on demand, allowing for the background free imaging, multiplexing, super-resolution, and contrast enhancement through both optical and acoustic modulation. This article will demonstrate how to synthesize optically-triggerable, lipid shell PFCnDs utilizing probe sonication, create polyacrylamide phantoms to characterize the nanodroplets, and acoustically modulate the PFCnDs after ODV to improve contrast.

Optically activated perfluorocarbon nanodroplets show promise in imaging applications outside of the vascular system. This article will demonstrate how to synthesize these particles, crosslink polyacrylamide phantoms, and modulate the droplets acoustically to enhance their signal.

Formulierung und akustische Modulation von optisch verdampften Perfluorcarbon-Nanotröpfchen

Microbubbles are the most commonly used imaging contrast agent in ultrasound. However, due to their size, they are limited to vascular compartments. These ...