Nous tenons à remercier le Dr Manfred Kriechbaum de Hecus XRS et l&#39;Institut de biophysique et de la recherche nanosystèmes à l&#39;Académie autrichienne des sciences à Graz, en Autriche. LL et XW ont été en partie soutenue par US Department of Energy, sous NERI-C sentence n ° DE-FG07-07ID14889, et US Nuclear Regulatory Commission, en vertu de l&#39;adjudication N º CNRC-38-08-950. L&#39;instrument SWAXS est également soutenue en partie par US Department of Energy, sous sentence n ° DE-NE0000325.

1. Préparation de l&#39;échantillon <ol><li> Utilisez une aiguille pour enlever une partie de l&#39;échantillon du récipient d&#39;échantillon. * </li><li> Utilisez l&#39;aiguille pour remplir le capillaire (diamètre maximum de 2,2 mm) avec l&#39;échantillon. Le capillaire doit être rempli entre 2 et 3 cm du fond. </li><li> Fermez le capillaire par fondre la cire sur sa pointe. </li><li> Dévisser le porte-échantillon vide du système. </li><li> Prenez le capillaire par l&#39;extrémité fondue (fin de la cire) et insérer l&#39;extrémité non fondu dans le porte-échantillon. </li><li> Placez le support dans le système et la vis en place. </li></ol> * Les échantillons solides (y compris les échantillons de poudre) peut être directement placé dans le porte-échantillon (pas nécessaire capillaire), tandis que les échantillons liquides doivent être placés dans un capillaire. Démarrage de la machine SWAXS 2. Procédure de démarrage à froid Source <ol><li> Fermez l&#39;obturateur en appuyant sur le bouton de l&#39;obturateur de sécurité. </li><li> Coupez l&#39;alimentation principale«ON» en appuyant sur le bouton vert. </li><li> Vérifiez l&#39;état d&#39;urgence &quot;Shut-Off&quot; est dans la position étendue. </li><li> Mettez l&#39;appareil sous tension en appuyant sur le bouton vert. </li><li> Vérifier que le voyant de verrouillage s&#39;allume en vert, indiquant tous les verrouillages sont ok. </li><li> Attendez que l&#39;écran tactile à charger. Une fois qu&#39;il a chargé, appuyez sur &quot;R6&quot; dans le menu. </li><li> Tournez la clé à la veille position &quot;ON&quot;. </li><li> Tournez la clé à rayons X pour la position &quot;ON&quot;. Le rouge aux rayons X de la lumière devrait s&#39;allumer. </li><li> Attendez que l&#39;écran tactile pour lire les niveaux d&#39;attente (30 kV et 0,4 mA). </li><li> Tournez la clé de sécurité veille à la position «OFF». Appuyez sur la touche &quot;Cycle Start&quot; en bas de l&#39;écran tactile. </li><li> Attendez que l&#39;écran tactile pour lire la puissance nominale (50 kV et 1 mA). Si un niveau de puissance différent est souhaité, entrez dans l&#39;écran de configuration en appuyant sur «R4» sur l&#39;écran tactile et d&#39;entrer les paramètres souhaités. </li></ol> 3. Procédure refroidisseur <ol><li> Tournez le pouvoir switch à la position «ON» sur le panneau de contrôle de la température. Un voyant de contrôle et l&#39;affichage LED de la température s&#39;allume. </li><li> Tournez le commutateur d&#39;alimentation sur la position &quot;ON&quot; sur le refroidisseur. </li><li> Attendez jusqu&#39;à ce que la température affichée est &quot;OFF&quot;. Ceci est en mode veille. </li><li> Appuyez et maintenez enfoncé le bouton Enter (symbole de retour) pendant environ 4 secondes ou jusqu&#39;à ce que l&#39;affichage de température indique une température réelle. </li><li> Pour modifier la température, appuyez sur le haut ou flèche vers le bas. La valeur de consigne sera montré sur l&#39;affichage de la température pendant environ 8 secondes avant qu&#39;il ne retourne à la valeur réelle. </li><li> Appuyez sur la touche ENTER une fois la valeur désirée s&#39;affiche. Cela permettra de stocker cette valeur. </li><li> Attendez que la température réelle atteint la valeur de consigne souhaitée. </li></ol> REMARQUE: Si, à un moment où l&#39;erreur &quot;E 01&quot; apparaît sur ​​l&#39;affichage de la température, suivez les instructions d&#39;arrêt du refroidisseur, remplir à nouveau bain en versant de l&#39;eau purifiée dans le réservoir de fill, puis effectuer la procédure de démarrage refroidisseur à nouveau. 4. Arrêt du refroidisseur <ol><li> Appuyez sur &quot;enter&quot; pour environ 4 sec. </li><li> Mettez l&#39;interrupteur d&#39;alimentation à la position «OFF». </li></ol> 5. Mise sous tension du vide <ol><li> S&#39;assurer que la porte vide est maintenu en place. </li><li> Tournez le vide 1 et 2 boutons pour les positions &quot;ON&quot;. </li><li> Attendez jusqu&#39;à ce que la jauge à vide VAP5 lit un niveau de vide inférieur à 1,5 mbar. </li></ol> 6. Configuration du système de détection <ol><li> Pour configurer la pression du gaz, vérifiez le manomètre principal de la valve de réduction est d&#39;au moins 10 bars. </li><li> Ouvrez la vanne principale. </li><li> Ouvrez lentement la valve de pression de fonctionnement jusqu&#39;à ce que la pression atteint 8 bars à la jauge de pression de fonctionnement. </li><li> Ouvrez la seconde valve principale </li><li> Pour régler le débit de gaz, ouvrez la soupape principale en tournant le bouton vers la position verticale sur le panneau de contrôle de gaz. </li><li> Régler le débittaux avec le pointeau jusqu&#39;à ce que le manomètre indique environ 8 bars. </li><li> Mettez l&#39;interrupteur d&#39;alimentation principale en position &quot;ON&quot;. La lumière LED doit s&#39;allumer. </li><li> Pour régler la haute tension, activez la haute tension en tournant le sélecteur sur la position &quot;ON&quot;. La lumière LED doit s&#39;allumer. </li><li> Tourner lentement le bouton de commande de tension jusqu&#39;à 3.5kV environ est atteint sur la jauge. NE PAS DEPASSER 4 kV. </li></ol> 7. Étalonnage <ol><li> Les étapes expérimentales ci-dessus sont effectuées sur un échantillon de pics connus. </li><li> 3,3 ASA est utilisé pour obtenir le graphique de l&#39;intensité par rapport à chaîne avec 5 sommets. </li><li> Allez à Menu → Options → Entrez les canaux et les intensités correspondantes. </li><li> Aller à la ASA3, &quot;TPF&quot; premier onglet. </li><li> Changer le filtre, en haut à gauche 1 mm Nickel (filtre de faisceau). </li><li> Régler la position 32.000 (28.000 à 32.000 gamme, zone critique autour de 28.000) → allez à la poste, faire le vide sûr, c&#39;est inférieur à 5 mbar, assurez-vous que le détecteur est raisonnableding entre 1k-10K. </li><li> Course à 10 sec pour environ 52 coups par seconde. </li><li> Changer les fenêtres de l&#39;énergie, (ou de mesurer la fenêtre d&#39;énergie avec l&#39;échantillon seulement, non compris le boîtier de filtre). Régler la course finale pendant 10 sec. </li><li> Trouver l&#39;emplacement du faisceau primaire (233 canal par exemple). </li><li> Pour d&#39;autres essais, changement de filtre à nouveau pour 2 Tungstène mm (W, faisceau bouchon) et à partir de 30.000. </li><li> Trouvez l&#39;emplacement de tous les autres pics des échantillons. </li><li> Utiliser ImageJ-macro logiciel pour calibrer SAXS - les transitions à partir de l&#39;intensité par rapport à l&#39;intensité de canal par rapport à q (ou de l&#39;espacement d) (figure 1). </li><li> Utiliser ImageJ-macro logiciel pour calibrer WAXS - les transitions à partir de l&#39;intensité par rapport à l&#39;intensité de canal par rapport à q (ou de l&#39;espacement d) (figure 2). </li></ol> 8. Procédure logiciel <ol><li> Utilisez le logiciel ASA 3.3, pour recueillir les données en direct: Live Data → → TPF onglet Enregistrer le fichier. </li><li> Pour EasySWAXS, cliquez sur onglet Paramètres du périphérique. </li><li> Saisir le &quot;a&quot;, lambda, et d valeurs, ainsi que le centre de gravité. </li><li> Sélectionnez Collimation Point. </li><li> Sélectionnez le type de particules globulaires (sauf si c&#39;est un connu pour être une tige de particules en forme de plat). </li><li> Cliquez sur l&#39;onglet Guinier-Terrain. Un je vs q 2 graphique s&#39;affiche. </li><li> Faites glisser les lignes verticales pour entourer une partie de la pente à peu près linéaire. </li><li> Au bas de l&#39;écran, une valeur de R est affiché. A côté de cela, il ya une exigence de validation. </li><li> Continuer à faire glisser les lignes verticales se rapprocher jusqu&#39;à ce que la valeur de validation est comprise entre 1 et 2. Ceci est subjective. La valeur R ou rayon de giration, obtenu est une estimation. </li></ol> 9. Procédure d&#39;arrêt source <ol><li> Vérifier l&#39;innocuité et obturateurs rapides sont fermés. </li><li> Tournez la clé à la veille position &quot;ON&quot;. </li><li> Attendez que l&#39;écran tactile pour lire les niveaux de veille. </li><li> Presse R5 sur le touchscreen. </li><li> Réduire le courant à 0 A. </li><li> Réduire la tension à 0 A. </li><li> Tournez la clé à rayons X pour la position «OFF». </li><li> Coupez l&#39;alimentation principale &quot;OFF&quot; en appuyant sur le bouton d&#39;arrêt rouge. </li></ol>

<ol>
	<li>Bernad&#243;, P., Blackledge, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+biology:+Proteins+in+dynamic+equilibrium.">Structural biology: Proteins in dynamic equilibrium.</a> Nature. 468, 1046-1048 (2010).</li><li>Zhang, F., Skoda, M. W. A., Jacobs, R. M. J., Martin, R. A., Martin, C. M., Schreiber, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Interactions+Studied+by+SAXS:+Effect+of+Ionic+Strength+and+Protein+Concentration+for+BSA+in+Aqueous+Solutions.">Protein Interactions Studied by SAXS: Effect of Ionic Strength and Protein Concentration for BSA in Aqueous Solutions.</a> J. Phys. Chem. B. 111, 251-259 (2007).</li><li>Maranas, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+environment+on+local+dynamics+of+macromolecules.">The effect of environment on local dynamics of macromolecules.</a> Current Opinion in Colloid &amp; Interface Science. 12, 29-42 (2007).</li><li>Stribeck, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> X-Ray Scattering of Soft Matter. , (2007).</li><li>Mertens, H. D., Svergun, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+characterization+of+proteins+and+complexes+using+small-angle+X-ray+solution+scattering.">Structural characterization of proteins and complexes using small-angle X-ray solution scattering.</a> J. Struct. Biol. 172 (1), 128 (2010).</li><li>Svergun, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-angle+X-ray+and+neutron+scattering+as+a+tool+for+structural+systems+biology.">Small-angle X-ray and neutron scattering as a tool for structural systems biology.</a> Biol. Chem. 391 (7), 737 (2010).</li><li>Putnam, C. D., Hammel, M., Hura, G. L., Tainer, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+solution+scattering+(SAXS)+combined+with+crystallography+and+computation:+defining+accurate+macromolecular+structures,+conformations+and+assemblies+in+solution.">X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution.</a> Quat. Rev. Biophys. 40, 191-285 (2007).</li><li>Bonini, M., Fratini, E., Baglioni, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SAXS+study+of+chain-like+structures+formed+by+magnetic+nanoparticles.">SAXS study of chain-like structures formed by magnetic nanoparticles.</a> Materials Science &amp; Engineering C-Biomimetic and Supramolecular Systems. 27 (5-8), 1377-1381 (2007).</li><li>Falletta, E., Ridi, F., Fratini, E., Vannucci, C., Canton, P., Bianchi, S., Castelvetro, V., Baglioni, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tri-block+copolymer+templated+synthesis+of+gold+nanostructures.">A tri-block copolymer templated synthesis of gold nanostructures.</a> Journal of Colloid and Interface Science. 357 (1), 88-94 (2011).</li><li>Glatter, O., Scherf, G., Schillen, K., Brown, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+Poly(ethylene+oxide)+Poly(propylene+oxide)+Triblock+Copolymer+(EO(27)-PO39-EO(27))+in+Aqueous-Solution.">Characterization of a Poly(ethylene oxide) Poly(propylene oxide) Triblock Copolymer (EO(27)-PO39-EO(27)) in Aqueous-Solution.</a> Macromolecules. 27 (21), 6046-6054 (1994).</li><li>Mittelbach, R., Glatter, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+structure+analysis+of+small-angle+scattering+data+from+polydisperse+colloidal+particles.">Direct structure analysis of small-angle scattering data from polydisperse colloidal particles.</a> Journal of Applied Crystallography. 31, 600-608 (1998).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

La procédure comparative du système SWAXS permet de nombreuses variables à déterminer à partir de l&#39;analyse expérimentale. Les paramètres qui sont obtenus à partir de l&#39;analyse peut être utilisée à différentes fins selon l&#39;échantillon et montage expérimental. SAXS fournit des informations sur la nano-échelle de la taille et de la forme de l&#39;objet, alors que WAXS se concentre sur la structure atomique et micro-échelle (par exemple, réseau moléculaire, la symétrie dimensions de ...

<table border="1"><tbody><tr><td> Nom des produits </td><td> Entreprise </td></tr><tr><td> Le System3 petites et grand-angle X-ray Scattering (SWAXS) Caméra </td><td> Hecus XRS et IBN, Graz, Autriche </td></tr><tr><td> GNOM </td><td> ATSAS 2,3 paquet par D. Svergun EMBL-Hambourg </td></tr></tbody></table>

small and wide angle x-ray scattering studies of biological macromolecules in solution

Dans cet article, les petites et Wide Angle X-ray Scattering (SWAXS) l&#39;analyse des macromolécules est démontrée par l&#39;expérimentation. SWAXS est une technique dans laquelle les rayons X sont diffusés élastiquement par un échantillon non homogène dans la gamme nm aux petits angles (typiquement de 0,1 à 5 °) et des angles larges (typiquement&gt; 5 °). Cette technique fournit des informations sur la forme, la taille et la distribution des macromolécules, les distances caractéristiques de matériaux partiellement ordonnés, tailles de pores et surface-volume élevé. Petit angle X-ray Scattering (SAXS) est capable de fournir des informations de structure de macromolécules entre 1 et 200 nm, tandis que le grand angle X-ray Diffraction (WAXS) peut résoudre encore plus faible espacement entre les échantillons de Bragg 0,33 nm et 0,49 nm sur la base de l&#39; configuration spécifique du système et le détecteur. La distance est déterminée à partir de la loi de Bragg et est fonction de la longueur d&#39;onde et angle d&#39;incidence. Dans une expérience SWAXS, les matériaux peuvent être solidesou liquide, ou contenir des solides, liquides ou gazeux domaines (dites particules) de la même matière ou d&#39;une autre dans une combinaison quelconque. SWAXS applications sont très larges et comprennent des colloïdes de tous types: métaux, composites, ciment, pétrole, les polymères, les plastiques, les protéines, les aliments et les produits pharmaceutiques. Pour des échantillons solides, l&#39;épaisseur est limitée à environ 5 mm. L&#39;utilisation d&#39;un instrument de laboratoire à base de SWAXS est détaillé dans cet article. Avec les logiciels disponibles (par exemple, GNOM-ATSAS 2,3 paquet par D. Svergun EMBL-Hambourg et logiciels EasySWAXS) pour le système SWAXS, une expérience peut être menée afin de déterminer certains paramètres d&#39;intérêt pour l&#39;échantillon donné. Un exemple d&#39;une expérience macromolécule biologique est l&#39;analyse de 2% en poids de lysozyme dans un tampon aqueux à base d&#39;eau qui peut être choisi et préparé par diverses méthodes. La préparation de l&#39;échantillon respecte les directives ci-dessous dans la section Préparation des échantillons. Par l&#39;expérimentation SWAXS,importantes des paramètres structurels du lysozyme, par exemple, le rayon de giration, peut être analysée.

SAXS et WAXS tout peut fournir des informations de structure de l&#39;échantillon au travers des paramètres suivants: le rayon de giration granulométrie, et la forme, facteur de structure de la solution, la surface spécifique interne et la taille des pores, de type treillis et les dimensions, et la densité d&#39;électrons 1. SAXS et WAXS peut également être appliquée à l&#39;étude de 2 dynamique des protéines. Les informations de structure d&#39;expériences...

Watch this Scientific Journal Video about Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution at JoVE.com

Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution | Bioengineering | Video

Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

La démonstration de l&#39;angle très faible et large diffusion des rayons X (SWAXS) procédure est devenue contribué à l&#39;étude des macromolécules biologiques. Grâce à l&#39;utilisation de l&#39;instrumentation et des procédures de méthodes de préparation et d&#39;angle spécifiques, les données expérimentales de la SWAXS affiche la caractérisation atomique et nano-échelle de macromolécules.

Les petites et Wide Angle études diffraction des rayons X de macromolécules biologiques en solution

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique ...

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17.9K vues.  Rensselaer Polytechnic Institute. La démonstration de l'angle très faible et large diffusion des rayons X (SWAXS) procédure est devenue contribué à l'étude des macromolécules biologiques. Grâce à l'utilisation de l'instrumentation et des procédures de méthodes de préparation et d'angle spécifiques, les données expérimentales de la SWAXS affiche la caractérisation atomique et nano-échelle de macromolécules.

Vidéo: Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

La fabrication des électrochimique de l&#39;ADN des Biocapteurs pour la détection sans réactif d&#39;acides nucléiques, protéines et petites molécules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

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In situ TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

In situ TEM of Biological Assemblies in Liquid

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

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 ...

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.

Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

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Studying Soft-matter and Biological Systems over a Wide Length-scale from Nanometer and Micrometer Sizes at the Small-angle Neutron Diffractometer KWS-2

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. The instrument is optimized for the exploration of the wide momentum transfer Q range between 1x10-4 and 0.5 &#197;-1 by combining classical pinhole, focusing (with lenses), and time-of-flight (with chopper) methods, while simultaneously providing high-neutron intensities with an adjustable resolution. Because of its ability to adjust the intensity and the resolution within wide limits during the experiment, combined with the possibility to equip specific sample environments and ancillary devices, the KWS-2 shows a high versatility in addressing the broad range of structural and morphological studies in the field. Equilibrium structures can be studied in static measurements, while dynamic and kinetic processes can be investigated over time scales between minutes to tens of milliseconds with time-resolved approaches. Typical systems that are investigated with the KWS-2 cover the range from complex, hierarchical systems that exhibit multiple structural levels (e.g., gels, networks, or macro-aggregates) to small and poorly-scattering systems (e.g., single polymers or proteins in solution). The recent upgrade of the detection system, which enables the detection of count rates in the MHz range, opens new opportunities to study even very small biological morphologies in buffer solution with weak scattering signals close to the buffer scattering level at high Q.
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The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

La boucle artério (AV) dans un modèle Small Animal pour étudier l&#39;angiogenèse et du génie tissulaire vascularisé

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

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 ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

Structuration des protéines bioactives ou Peptides sur Hydrogel utilisant photochimie pour Applications biologiques

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior in such restrictive conditions. To address this issue, we have developed growth chambers for tip-growing plant cells that contain a series of narrow, micro-fabricated gaps (~1 &#181;m) in a poly-dimethylsiloxane (PDMS) substrate. This transparent material allows the user to monitor tip elongation processes in individual cells during microgap penetration by time-lapse imaging. Using this experimental platform, we observed morphological changes in pollen tubes as they penetrated the microgap. We captured the dynamic changes in the shape of a fluorescently labeled vegetative nucleus and sperm cells in a pollen tube during this process. Furthermore, we demonstrated the capability of root hairs and moss protonemata to penetrate the 1 &#181;m gap. This in vitro platform can be used to study how individual cells respond to physically constrained spaces and may provide insights into tip-growth mechanisms.

We describe a method to investigate the capability of tip-growing plant cells, including pollen tubes, root hairs, and moss protonemata, to elongate through extremely narrow gaps (~1 &#181;m) in a microfluidic device.

Développement de dispositifs microfluidiques pour étudier la capacité d’élongation de la pointe de croissance des cellules végétales dans des espaces très réduits

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior ...

Using Nanoplasmon-Enhanced Scattering and Low-Magnification Microscope Imaging to Quantify Tumor-Derived Exosomes

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes have the potential to serve as biomarkers for disease diagnosis and to monitor disease progression and treatment response. However, most exosome analysis procedures require exosome isolation and purification steps, which are usually time-consuming and labor-intensive, and thus of limited utility in clinical settings. This report describes a rapid procedure to analyze specific biomarkers on the outer membrane of exosomes without requiring separate isolation and purification steps. In this method, exosomes are captured on the surface of a slide by exosome-specific antibodies and then hybridized with nanoparticle-conjugated antibody probes specific to a disease. After hybridization, the abundance of the target exosome population is determined by analyzing low-magnification dark-field microscope (LMDFM) images of the bound nanoparticles. This approach can be easily adopted for research and clinical use to analyze membrane-associated exosome biomarkers linked to disease.

Clinical translation of exosome-derived biomarkers for diseased and malignant cells is hindered by the lack of rapid and accurate quantification methods. This report describes the use of low-magnification dark-field microscope images to quantify specific exosome subtypes in small volume serum or plasma samples.

Utilisation de la diffusion améliorée par Nanoplasmon et imagerie microscopique à faible grossissement pour quantifier les exosomes dérivés de tumeurs

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes ...

Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate structures&#160;while processing and histological observation provide limited information through cross-sectional imaging. Micro X-ray computed tomography (microCT) is an effective tool for obtaining three-dimensional information without damaging specimens. However, it shows limited efficiency in differentiating soft tissue parts. Use of contrast-enhancing agents, like phosphotungstic acid (PTA), can solve this problem by improving soft tissue contrast. We implemented microCT with PTA to investigate the human orbicularis retaining ligament (ORL), which is a delicate structure in the orbit area. In this method, harvested specimens are fixed in formalin, dehydrated in serial ethanol solutions, and stained with a PTA solution. After staining, microCT scanning, 3D reconstruction, and analysis are performed. Skin, ligaments, and muscles can be clearly visualized using this method. The specimen size and duration of staining are essential features of the method. The suitable specimen thickness was about 5&#8211;7 mm, above which the process was slowed, and the optimum duration was 5&#8211;7 days, below which an empty hole in the central area occasionally occurred. To maintain the location and direction of small pieces during cutting, sewing on the same region of each part is recommended. Furthermore, preliminary analyses of the anatomical structure are needed to correctly identify each piece. Parafilm can be used to prevent drying, but care should be taken to prevent specimen distortion. Our multidirectional observation showed that the ORL is composed of a multilayered meshwork of continuous plates, rather than thread-like fibers, as reported previously. These results suggest that microCT scanning with PTA is useful for examining specific compartments within complex structures of human tissue. It may be helpful in the analyses of cancer tissues, nerve tissues, and various organs, like the heart and liver.

Micro X-ray computed tomography is effective in obtaining three-dimensional information from undamaged human specimens but has limited success in observing soft tissues. The use of phosphotungstic acid contrast agent can resolve this issue. We implemented this contrast agent to examine human delicate fibromuscular tissues (the orbicularis retaining ligament).

Utilisation de la tomographie calculée par rayons X micro avec préparation d'acide phosphotungstic pour visualiser le tissu fibromusculaire humain

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate ...