Les auteurs remercient le Dr Michael J. Friedlander, directeur du Virginia Tech Carilion Research Institute, d’avoir encouragé nos efforts de recherche. Ce projet a été soutenu par des fonds de développement de S.M.M et D.F.K. et en partie par l’initiative Nano-Bio de l’Institute for Critical Technology and Applied Science de Virginia Tech.

1. Préparer les périphériques de capture d’affinité16
<ol><li>Nettoyer les E-puces de nitrure de silicium en les incubant dans 15 ml d’acétone pendant 2 min suivi de 15 ml de méthanol pendant 2 min(figure 1A). Laissez les copeaux sécher sous un flux d’air laminaire.</li>
	<li>Incuber les copeaux séchés sur une plaque d’agitation chauffée (sans agitation) pendant 1,5 heure à 150 °C, puis les laisser refroidir à température ambiante avant utilisation.</li>
	<li>Utilisez des seringues Hamilton pour composer des mélanges de lipides dans de petits tubes de verre afin de contenir 25 % de chloroforme, 55 % de DLPC (1,2-dilauroyl-phosphocholine) dans le chloroforme (1 mg/ml) et 20 % de lipides Ni-NTA (sel d’acide 1,2-dioleoyl-iminodiacétique-succinyl-nickel) dans le chloroforme (1 mg/ml) pour un volume total de 40 μl.</li>
	<li>Appliquer une partie aliquote de 1 μl du mélange sur chaque goutte de Milli-Q de 15 μl sur un morceau de Parafilm dans une boîte de Petri humide(figure 1B). Incuber des échantillons sur de la glace pendant au moins 60 min.</li>
</ol>2. Capturer des macromolécules17
<ol><li>Placer une puce électronique avec une entretoise intégrée de 150 nm sur un échantillon monocouche et incuber pendant 1 min(Figure 1C).</li>
	<li>Soulevez délicatement la puce de l’échantillon et ajoutez une aliquote de 3 μl de la protéine A marquée par His. Incuber pendant 1 min à température ambiante(figure 1D).</li>
	<li>Épongez l’excès de goutte à l’aide de Whatman #1 papier filtre et ajoutez immédiatement une aliquote de 3 μl de solution d’anticorps. Incuber pendant 1 min à température ambiante.</li>
	<li>Retirer l’excès de solution à l’aide d’une seringue Hamilton et ajouter immédiatement une partie aliquote de 1 μl de DLPs rotavirus (0,1 mg/ml) dans une solution tampon contenant 50 mM hepes (pH 7,5), 150 mM NaCl, 10 mM MgCl2 et 10 mM CaCl2. Incuber pendant au moins 2 min à température ambiante. La préparation des DLP a été décrite précédemment18.
</li>
</ol>3. Assemblez la chambre microfluidique et chargez le porte-échantillon in situ
<ol><li>Chargez la puce électronique humide contenant l’échantillon viral dans la pointe du porte-échantillon microfluidique. Décharger une seconde puce E plate pendant 1 min puis chargée sur le dessus de la puce d’entretoise(Figure 2, panneaux 1-5).</li>
	<li>Alternativement, pour augmenter le contraste des macromolécules biologiques, une tache de métaux lourds(par exemple 0,2% d’uranyl formate) peut être ajoutée aux échantillons humides avant de placer la deuxième puce électronique sur l’échantillon humide. Cependant, la puce électronique contenant l’échantillon devra être lavée avec de l’eau Milli-Q avant d’ajouter le réactif de contraste.</li>
	<li>Sandwich l’ensemble de l’ensemble ensemble pour former une enceinte scellée, maintenue en place mécaniquement à l’intérieur du support par 3 vis en laiton(Figure 2,panneaux 6-8).</li>
	<li>Après assemblage, la pointe du support est pompée à 10-6 Torr à l’aide d’une station de pompage à sec turbo-pompée avant de placer le support à l’intérieur du TEM.</li>
</ol>4. Imagerie dans un liquide à l’aide d’un microscope électronique à transmission
<ol><li>Chargez le porte-échantillon in situ dans un microscope électronique à transmission (FEI Company) équipé d’un filament LaB6 et fonctionnant à 120 kV.</li>
	<li>Allumez le filament TEM et ajustez la hauteur eucentrique de l’étage du microscope par rapport à l’échantillon en utilisant la fonction wobbler pour incliner l’échantillon de -15° à +15° d’avant en arrière dans la colonne. Cette procédure ajuste l’étage dans la direction z pour aider à tenir compte de l’épaisseur appropriée de la chambre à liquide. Cette étape garantit également qu’un grossissement précis est utilisé lors de l’enregistrement d’images.</li>
	<li>Enregistrez d’abord les images le long des bords et dans les régions d’angle de la chambre microfluidique. Ces zones contiennent généralement la solution la plus fine. Enregistrer des images d’échantillons dans des conditions de faible dose (1-3 électrons/Å2)à l’aide d’une caméra CCD. Utilisez des grossissements nominaux de 6 000X à 30 000X.</li>
	<li>Déterminez la valeur de défocalisation appropriée en vous concentrant sur le bord de la chambre fluidique. Utilisez une valeur de -1,5 μm pour enregistrer des images à un grossissement de 30 000X. Si une solution épaisse est rencontrée ou si l’agent de contraste n’est pas utilisé dans la préparation de l’échantillon, utilisez des valeurs de défocalisation plus élevées dans la plage de -2 à -4 μm.</li>
	<li>Pour s’assurer que la solution est contenue dans la chambre microfluidique tout au long des expériences, concentrez le faisceau d’électrons jusqu’à ce que les bulles se forment dans le liquide à l’intérieur de l’appareil.</li>
</ol>

<ol>
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L’auteur, Madeline J. Dukes, est une employée de Protochips, Inc.

Dans notre travail présenté, nous avons employé l’approche de capture d’affinité pour attacher le rotavirus DLPs à une plate-forme microfluidique. Cela a permis l’imagerie in situ de complexes macromoléculaires dans un microenvironnement liquide. L’approche de capture est importante en ce qui concerne d’autres techniques d’imagerie microfluidique, car elle localise les échantillons biologiques dans la fenêtre d’imagerie pour annuler les grands problèmes diffusifs qui survie...

<a href="https://app.jove.com/t/6605">This corrects</a> the article <a href="https://dx.doi.org/10.3791/50936">10.3791/50936</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/50936">In situ TEM of Biological Assemblies in Liquid</a>. The Representative Results and References sections were&#160;updated.

Erratum: In situ TEM of Biological Assemblies in Liquid

Un objectif commun des biologistes et des ingénieurs est de comprendre le fonctionnement interne des machines moléculaires. Les microscopes électroniques à transmission (TEMs) sont des instruments idéaux pour visualiser ces détails complexes à une résolution quasi atomique1-2. Afin de soutenir le système à vide poussé d’un TEM, les échantillons biologiques sont typiquement encastrés dans des films minces de glace vitreuse3,de sucres4,de sels de métaux lourds5,ou d’une combinaison de ceux-ci6. Par conséquent, les images d’échantillons incorporés peuvent ne révéler que des instantanés limités de processus dynamiques.
Des tentatives précoces pour maintenir les spécimens biologiques hydratés dans des chambres liquides environnementales ont été entreprises par Parsons et collègues utilisant des étapes de pompage différentielles. Des modèles de diffraction d’électrons de cristaux de catalase non souillés ont été enregistrés avec succès à une résolution de 3 Å dans un état hydraté7-8. En outre, des domaines lipidiques séparés en phase ont pu être examinés dans des membranes hydratées d’érythrocytes humains9-10. Cependant, le mouvement causé par la diffusion du liquide et son interférence avec le faisceau d’électrons, a entraîné une perte de résolution grave et d’autres expériences utilisant des échantillons biologiques n’ont pas été tentées jusqu’à récemment.
De nouveaux supports d’échantillons microfluidiques ont été introduits qui utilisent des micropuces semi-conductrices pour former une chambre environnementale micro-échelle. Ces dispositifs peuvent maintenir les échantillons dans un liquide pendant qu’ils sont positionnés dans une colonne TEM11-12. Cette percée technique dans l’imagerie TEM a permis aux chercheurs de visualiser, pour la première fois, des événements progressifs au niveau moléculaire13. Nous appelons cette nouvelle modalité « microscopie moléculairein situ » car les expériences peuvent maintenant être effectuées « à l’intérieur » de la colonne EM14-15. L’objectif global de cette méthode est d’imager les assemblages biologiques dans un liquide afin d’observer leurs comportements dynamiques à une résolution nanométrique. La raison d’être de la technique mise au point est d’enregistrer les observations en temps réel et d’examiner les nouvelles propriétés des machines biologiques en solution. Cette méthodologie élargit l’utilisation des TEMs à des fins plus larges en biologie cellulaire et moléculaire12-16.
Dans l’article vidéo actuel, nous présentons un protocole complet pour assembler et utiliser un support d’échantillon microfluidique disponible dans le commerce. Ces supports spécialisés utilisent des micropuces de nitrure de silicium produites avec des entretoises intégrées pour former une chambre liquide qui renferme des volumes infimes de solution. Des fenêtres minces et transparentes sont gravées dans les micropuces à des fins d’imagerie12. Nous démontrons l’utilisation appropriée d’un support microfluidique pour examiner les particules à double couche (DLPs) du rotavirus simien dans un liquide à l’aide d’une TEM. Pour s’assurer que les assemblages biologiques, tels que les DLP, ne diffusent pas rapidement sur de grandes distances lors de l’imagerie, nous utilisons l’approche Affinity Capture pour les attacher à la surface de la chambre microfluidique16. Cette étape de capture moléculaire présente un avantage majeur par rapport aux techniques alternatives d’imagerie des échantillons biologiques dans un liquide, car elle permet l’acquisition d’images qui seront utilisées pour les routines de traitement en aval. Cette étape de capture utilisée en conjonction avec l’imagerie microfluidique est unique à nos procédures17. Les lecteurs qui utilisent des applications de biologie structurale à l’aide de la TEM ou de chambres d’imagerie microfluidiques peuvent envisager l’utilisation de techniques de capture d’affinité lorsque les observations dynamiques au niveau moléculaire sont l’objectif final.

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		<col />
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		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:237px;">E-chips, spacer chip</td>
			<td style="width:183px;">Protochips, Inc.</td>
			<td style="width:245px;">EPB-52TBD</td>
			<td style="width:167px;">400 &#956;m x 50 &#956;m window</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:237px;">E-chip, top chip</td>
			<td style="width:183px;">Protochips, Inc.</td>
			<td style="width:245px;">EPT-45W</td>
			<td style="width:167px;">400 &#956;m x 50 &#956;m window</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Ni-NTA lipid</td>
			<td style="width:183px;">Avanti Polar Lipids</td>
			<td style="width:245px;">790404P</td>
			<td style="width:167px;">Powder form</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">DLPC (12:0) lipid</td>
			<td style="width:183px;">Avanti Polar Lipids</td>
			<td style="width:245px;">850335P</td>
			<td style="width:167px;">Powder form</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Volumetric flasks&#160;</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">20-812A; 20-812C</td>
			<td style="width:167px;">1 ml; 5 ml&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Hamilton Syringes</td>
			<td style="width:183px;">Hamilton Co.</td>
			<td style="width:245px;">80300, 80400</td>
			<td style="width:167px;">1-10 &#956;l; 1-25 &#956;l</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Whatman #1 filter paper</td>
			<td style="width:183px;">Whatman</td>
			<td style="width:245px;">1001 090</td>
			<td style="width:167px;">100 pieces, 90 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glass Petri dishes</td>
			<td style="width:183px;">Corning&#160;</td>
			<td style="width:245px;">70165-101</td>
			<td style="width:167px;">100 mm x 15 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glass Pasteur pipettes</td>
			<td style="width:183px;">VWR</td>
			<td style="width:245px;">14673-010</td>
			<td style="width:167px;">14673-010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glass culture tubes</td>
			<td style="width:183px;">VWR</td>
			<td style="width:245px;">47729-566</td>
			<td style="width:167px;">6 mm x 50 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Acetone</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">&#160;A11-1</td>
			<td style="width:167px;">1 L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Methanol</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">&#160;A412-1</td>
			<td style="width:167px;">1 L</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">Chloroform&#160;</td>
			<td style="width:183px;">Electron Microcopy Sciences</td>
			<td style="width:245px;">12550</td>
			<td style="width:167px;">100 ml&#160;</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">His-tagged Protein A</td>
			<td style="width:183px;">Abcam, Inc.</td>
			<td style="width:245px;">ab52953</td>
			<td style="width:167px;">10 mg</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">Milli-Q water system</td>
			<td style="width:183px;">EMD Millipore Corp.</td>
			<td style="width:245px;">Z00QSV001</td>
			<td style="width:167px;">Ultrapure Water</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">HEPES</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">BP310-500</td>
			<td style="width:167px;">500 g</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">Equipment&#160;</td>
			<td style="width:183px;"></td>
			<td style="width:245px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">Poseidon In situ specimen holder</td>
			<td style="width:183px;">Protochips, Inc.</td>
			<td style="width:245px;"></td>
			<td>&#160;FEI compatible</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">FEI Spirit BioTwin TEM</td>
			<td style="width:183px;">FEI Co.</td>
			<td style="width:245px;"></td>
			<td>120 kV</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">Eagle 2k HS CCD camera</td>
			<td style="width:183px;">FEI Co.</td>
			<td style="width:245px;"></td>
			<td>10&#160;&#197;/pixel sampling at 30,000X</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:237px;">Gatan 655 Dry pump station</td>
			<td style="width:183px;">Gatan, Inc.</td>
			<td style="width:245px;"></td>
			<td>Pump holder tip to 10-6&#160;range</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:237px;">PELCO easiGlow, glow discharge unit</td>
			<td style="width:183px;">Ted Pella, Inc.</td>
			<td style="width:245px;"></td>
			<td>&#160;Negative polarity mode</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Isotemp heated stir plate</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;"></td>
			<td>Heat to 150 &#186;C for 1.5 hr</td>
		</tr>
	</tbody>
</table>

in situ tem of biological assemblies in liquid

Les chercheurs utilisent régulièrement des microscopes électroniques à transmission (TEMs) pour examiner des entités biologiques et évaluer de nouveaux matériaux. Ici, nous décrivons une application supplémentaire pour ces instruments: la visualisation d’assemblages viraux dans un environnement liquide. Cette méthode passionnante et nouvelle de visualisation des structures biologiques utilise un porte-spécimen à base de microfluidique récemment développé. Notre article vidéo montre comment assembler et utiliser un support microfluidique pour imager des spécimens liquides dans un TEM. En particulier, nous utilisons des particules à double couche (DLP) du rotavirus simien comme système modèle. Nous décrivons également les étapes pour recouvrir la surface de la chambre liquide de biofilms d’affinité qui attachent les DLP à la fenêtre de visualisation. Cela nous permet d’imager les assemblages d’une manière adaptée à la détermination de la structure 3D. Ainsi, nous présentons un premier aperçu des particules subvirales dans un environnement liquide natif.

Des images représentatives de DLP dans un liquide à l’aide de puces électroniques qui ont été déchargées par lueur(figure 3A)montrent moins de DLP dans une zone de visualisation donnée, probablement en raison de la diffusion, par rapport aux DLP qui sont enrichis sur des dispositifs de capture d’affinité(Figure 3B). L’ajout de formate d’uranyle dans la chambre d’imagerie améliore le contraste de l’échantillon et donc la visibilité des DLP individuels en solution<s...

Watch this Scientific Journal Video about In situ TEM of Biological Assemblies in Liquid at JoVE.com

In situ TEM of Biological Assemblies in Liquid

Nous décrivons ici une procédure pour imager des complexes viraux dans un liquide à une résolution nanométrique à l’aide d’un microscope électronique à transmission.

In situ TEM d’assemblages biologiques dans un liquide

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

in-situ-tem-of-biological-assemblies-in-liquid

Research

JoVE Journal

Bioengineering

In situ TEM of Biological Assemblies in Liquid

9.9K vues.  Protochips, Inc.. Nous décrivons ici une procédure pour imager des complexes viraux dans un liquide à une résolution nanométrique à l’aide d’un microscope électronique à transmission.

Vidéo: In situ TEM of Biological Assemblies in Liquid

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

L&#39;extraction des lipides cellulaires pour ciblées dilution isotopique stable analyse de spectrométrie de masse chromatographie liquide-

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Recherche

Bio-ingénierie

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Quantification de Global diastolique fonction de l&#39;analyse basée sur la modélisation cinématique de transmitral débit via le formalisme de remplissage diastolique paramétrée

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 &#176;C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.

Here we present a protocol to synthesize novel, high-aspect ratio biocomposites under biological conditions and in liquid media. The biocomposites scale from nanometers to micrometers in diameter and length, respectively. Copper nanoparticles (CNPs) and copper sulfate combined with cystine are the key components for the synthesis.

Génération des solutions évolutives de métal de très haute-Aspect Ratio nanocomposites dans un milieu liquide biologique

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

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

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

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

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

En cartographie in situ des propriétés mécaniques des biofilms par Microrhéologie de particules de suivi

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

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Magnétique alternatif microgels de gélatine hybride sensible au champ de libération contrôlée de médicaments

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

Une nouvelle technique de production et d&#39;observation de chimiluminescence en milieu biologique

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Automatisé de robotique Liquid Handling assemblage de dispositifs modulaires ADN

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Un<em&gt; In vitro</em&gt; Orgue Culture Modèle de la murine disque intervertébral

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Synthèse de cristaux liquides biocompatibles mousses élastomères comme échafaudages cellulaires pour les cultures de cellules spatiales 3D

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

Dispositifs électriques et magnétiques de champ pour la stimulation des tissus biologiques

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

Methods Article

In situ TEM d’assemblages biologiques dans un liquide