Vorremmo ringraziare il Dott. Manfred Kriechbaum di XRS Hecus e l&#39;Istituto di Biofisica e della Ricerca Nanosystems presso l&#39;Accademia Austriaca delle Scienze a Graz, in Austria. LL e XW sono stati sostenuti in parte da US Department of Energy, sotto NERI-C Award No. DE-FG07-07ID14889, e US Nuclear Regulatory Commission, sotto Award n NRC-38-08-950. Lo strumento SWAXS è supportato anche in parte da US Department of Energy, sotto Award No. DE-NE0000325.

1. Preparazione del campione <ol><li> Utilizzare un ago per rimuovere una porzione del campione dal contenitore. * </li><li> Utilizzare l&#39;ago per riempire il capillare (diametro massimo di 2,2 mm) con il campione. Il capillare deve essere riempito tra 2 e 3 cm dal fondo. </li><li> Chiudere il capillare di fusione della cera sulla punta. </li><li> Svitare il supporto del campione vuoto dal sistema. </li><li> Prendere la capillare entro la fine fuso (fine cera) e inserire il non-fuso estremità nel supporto del campione. </li><li> Posizionare il supporto nel sistema e avvitare. </li></ol> * Campioni solidi (compresi campioni di polvere) può essere direttamente collocato nel supporto del campione (non necessario capillare), mentre campioni liquidi deve essere posto in un capillare. Start-Up della Macchina SWAXS 2. Fonte procedura di avvio a freddo <ol><li> Chiudere l&#39;otturatore premendo il pulsante di scatto di sicurezza. </li><li> Spegnere l&#39;interruttore principale&quot;ON&quot;, premendo il tasto verde. </li><li> Verificare che il pulsante di emergenza &quot;Shut-Off&quot; è in posizione estesa. </li><li> Accendere l&#39;alimentazione principale premendo il tasto verde. </li><li> Verificare che la spia di blocco LED è verde e indica tutti gli interblocchi sono ok. </li><li> Attendere che il touch screen da caricare. Una volta caricato, premere il tasto &quot;R6&quot; nel menu. </li><li> Ruotare il tasto Standby sulla posizione &quot;ON&quot;. </li><li> Ruotare la radiografia chiave in posizione &quot;ON&quot;. La radiografia luce rossa deve accendersi. </li><li> Attendere che il touch screen per leggere i livelli di standby (30 kV e 0,4 mA). </li><li> Girare la chiave di standby di sicurezza nella posizione &quot;OFF&quot;. Premere il tasto &quot;Cycle Start&quot; pulsante nella parte inferiore del touch screen. </li><li> Attendere che il touch screen per leggere la potenza nominale (50 kV e 1 mA). Se un livello di potenza diversa si desidera, accedere alla schermata di configurazione premendo &quot;R4&quot; sul touch screen e inserendo le impostazioni desiderate. </li></ol> 3. Chiller Procedura <ol><li> Ruotare la swi alimentazionetch in posizione &quot;ON&quot; sul pannello di controllo della temperatura. Una luce di controllo e il display della temperatura a LED si accende. </li><li> Portare l&#39;interruttore di alimentazione in posizione &quot;ON&quot; sul refrigeratore. </li><li> Attendere fino a quando il display della temperatura indica &quot;OFF&quot;. Questa è la modalità di standby. </li><li> Premere e tenere premuto il pulsante di invio (simbolo di ritorno) per circa 4 secondi o fino a quando il display della temperatura indica una temperatura effettiva. </li><li> Per modificare la temperatura, premere la freccia su o freccia giù. Il valore impostato viene visualizzato sul display della temperatura per circa 8 secondi prima che possa ritornare al valore effettivo. </li><li> Premere il tasto ENTER una volta il valore di riferimento desiderato. Questo salverà il valore. </li><li> Attendere fino a quando la temperatura effettiva raggiunge il valore impostato desiderato. </li></ol> NOTA: Se in qualsiasi momento l&#39;errore &quot;E 01&quot; compare sul display della temperatura, seguire le istruzioni refrigeratore arresto, l&#39;unità di ricarica bagno versando acqua purificata nel fil serbatoiol, e quindi eseguire la procedura di avvio di nuovo refrigeratore. 4. Arresto di Chiller <ol><li> Premere &quot;enter&quot; per circa 4 secondi. </li><li> Portare l&#39;interruttore di alimentazione in posizione &quot;OFF&quot;. </li></ol> 5. Accensione del vuoto <ol><li> Assicurare la porta vuoto è fissato in posizione. </li><li> Girare il vuoto 1 e 2 manopole per i posizioni &quot;ON&quot;. </li><li> Attendere che il VAP5 vacuometro legge un livello di vuoto inferiore a 1,5 mbar. </li></ol> 6. Rivelatore di impostazione del sistema <ol><li> Per impostare la pressione del gas, verificare che il manometro principale della valvola di riduzione è di almeno 10 bar. </li><li> Aprire la valvola principale. </li><li> Aprire lentamente la valvola di pressione di esercizio fino a quando la pressione raggiunge 8 bar sul manometro di esercizio. </li><li> Aprire la seconda valvola principale </li><li> Per regolare la portata del gas, aprire la valvola principale della pressione ruotando la manopola in posizione verticale sul pannello di controllo del gas. </li><li> Regolare il flussotariffa con la valvola a spillo fino a quando l&#39;indicatore di legge circa 8 bar. </li><li> Portare l&#39;interruttore di alimentazione principale in posizione &quot;ON&quot;. La luce LED si illumina. </li><li> Per regolare l&#39;alta tensione, attivare l&#39;alta tensione portando l&#39;interruttore in posizione &quot;ON&quot;. La luce LED si illumina. </li><li> Ruotare lentamente la manopola di tensione di controllo fino a circa 3.5kV viene raggiunto sul manometro. NON SUPERARE 4kV. </li></ol> 7. Taratura <ol><li> Le fasi sperimentali sopra vengono eseguite con un campione di picchi noti. </li><li> ASA 3.3 è utilizzato per ottenere il grafico di intensità vs canale con 5 picchi. </li><li> Passare a Menu → Opzioni → Inserisci canali e intensità corrispondenti. </li><li> Vai a ASA3, scheda &quot;TPF&quot; prima. </li><li> Sostituire il filtro, in alto a sinistra 1 millimetro Nichel (fascio di filtro). </li><li> Imposta la posizione 32.000 (intervallo 28.000 a 32.000, regione critica circa 28.000) → vai a posizione, creare il vuoto che è inferiore a 5 mbar, assicurarsi che il rilevatore è ragioneding tra 1k-10K. </li><li> Eseguire per 10 secondi per circa 52 impulsi al secondo. </li><li> Cambiare le finestre di energia, (o misurare la finestra di energia con l&#39;unico campione, non compreso il caso del filtro). Impostare la corsa finale per 10 sec. </li><li> Individuare la posizione del fascio primario (233 canale per esempio). </li><li> Per le altre piste, sostituzione del filtro di nuovo a 2 Tungsten mm (W, fascio tappo), a partire da 30.000. </li><li> Trovare la posizione di tutti gli altri picchi dei campioni. </li><li> Utilizzare ImageJ-macro software per calibrare SAXS - loro transizione da intensità vs canale di intensità vs q (o spaziatura d) (Figura 1). </li><li> Utilizzare ImageJ-macro software per calibrare WAXS - loro transizione da intensità vs canale di intensità vs q (o spaziatura d) (Figura 2). </li></ol> 8. Software Procedura <ol><li> Utilizzare il software ASA 3.3, per la raccolta dei dati in tempo reale: dati in diretta → TPF scheda → Salva file. </li><li> Per EasySWAXS, fare clic sulla scheda Impostazioni periferica. </li><li> Immettere la &quot;a&quot;, lambda, e valori d, così come il centro di gravità. </li><li> Selezionare Punto di collimazione. </li><li> Selezionare il tipo di particella globulare (a meno che non si tratta di un noto per essere una canna di appartamento a forma di particelle). </li><li> Fare clic sulla scheda Guinier-Plot. Un I vs q 2 grafico verrà visualizzato. </li><li> Trascinare le linee verticali per circondare un tratto di pendenza pressoché lineare. </li><li> Nella parte inferiore della schermata, un valore di R viene visualizzato. Accanto a questo vi è un requisito di convalida. </li><li> Continuare a trascinare le linee verticali avvicinare finché il valore di convalida è tra 1 e 2. Questo è soggettiva. Il valore di R, o raggio della rotazione, che si ottiene è una stima. </li></ol> 9. Fonte Procedura di arresto <ol><li> Verificare la sicurezza e persiane veloci sono chiuse. </li><li> Ruotare il tasto Standby sulla posizione &quot;ON&quot;. </li><li> Attendere che il touchscreen per leggere i livelli di standby. </li><li> Premere R5 sulla touchscreen. </li><li> Ridurre la corrente a 0 A. </li><li> Ridurre la tensione a 0 A. </li><li> Ruotare la radiografia a chiave in posizione &quot;OFF&quot;. </li><li> Spegnere l&#39;alimentazione principale su &quot;OFF&quot; premendo il pulsante rosso spento. </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>

Nessun conflitto di interessi dichiarati.

La procedura comparativa del sistema SWAXS consente numerose variabili che devono essere determinati da analisi sperimentale. I parametri che vengono raggiunti dalla analisi può essere utilizzato per scopi diversi a seconda del campione e la configurazione sperimentale. SAXS fornisce informazioni su scala nanometrica dimensioni e la forma dell&#39;oggetto, mentre WAXS si concentra sulla struttura atomica e micro-scala (ad esempio reticolo molecolare, cellulare simmetria unità di misura). Più specificamente, ...

<table border="1"><tbody><tr><td> Nome dei prodotti </td><td> Azienda </td></tr><tr><td> Il System3 piccole e Wide-Angle X-Ray Scattering (SWAXS) Camera </td><td> Hecus XRS e IBN, Graz, Austria </td></tr><tr><td> GNOM </td><td> ATSAS 2,3 pacchetto D. Svergun EMBL-Amburgo </td></tr></tbody></table>

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

In questo lavoro, piccole e Wide Angle X-ray Scattering (SWAXS) analisi delle macromolecole è dimostrata attraverso la sperimentazione. SWAXS è una tecnica in cui i raggi X sono elasticamente sparsi da un campione disomogeneo nel nm-gamma a piccoli angoli (tipicamente 0.1 - 5 °) e angoli di larghezza (in genere&gt; 5 °). Questa tecnica fornisce informazioni circa la forma, le dimensioni e la distribuzione di macromolecole, distanze caratteristici di materiali parzialmente ordinati, dimensioni dei pori, e superficie-volume di rapporto. Piccolo angolo X-ray Scattering (SAXS) è in grado di fornire informazioni strutturali di macromolecole tra 1 e 200 nm, mentre Wide Angle X-ray Scattering (WAXS) può risolvere ancora minore spaziatura Bragg di campioni tra 0,33 e 0,49 nm nm basata sulla configurazione del sistema specifico e rivelatore. La distanza è determinata dalla legge di Bragg e dipende dalla lunghezza d&#39;onda e angolo incidente. In un esperimento SWAXS, i materiali possono essere solidio liquido e può contenere solidi, liquidi o gassosi domini (cosiddette particelle) dello stesso materiale o di un altro in qualsiasi combinazione. Applicazioni SWAXS sono molto ampie e comprendono colloidi di tutti i tipi: metalli, materiali compositi, cemento, olio, polimeri, materie plastiche, proteine, alimenti e prodotti farmaceutici. Per campioni solidi, lo spessore è limitata a circa 5 mm. L&#39;utilizzo di un laboratorio strumento basato SWAXS è dettagliata in questo articolo. Con il software a disposizione (ad esempio, GNOM-ATSAS 2,3 pacchetto D. Svergun EMBL-Amburgo e software EasySWAXS) per il sistema SWAXS, un esperimento può essere condotto per determinare alcuni parametri di interesse per il campione dato. Un esempio di un esperimento macromolecola biologica è l&#39;analisi del 2% in peso di lisozima in una base di acqua tampone acquoso che possono essere scelti e preparati con numerosi metodi. La preparazione del campione segue le istruzioni riportate nella sezione Preparazione dei campioni. Attraverso la sperimentazione SWAXS,importanti parametri strutturali di lisozima, ad esempio, il raggio di girazione, possono essere analizzati.

SAXS e WAXS complessivamente possono fornire informazioni strutturali del campione attraverso i seguenti parametri: il raggio di girazione, dimensione delle particelle e la forma, la struttura fattore soluzione, specifica superficie interna e la dimensione dei pori, tipo lattice e dimensione e densità elettronica 1. SAXS e WAXS può essere applicato anche allo studio della dinamica delle proteine ​​2. L&#39;informazione strutturale di esperimenti SWAXS viene ottenu...

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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 dimostrazione dell&#39;angolo piccolo e grande X-ray scattering (SWAXS) procedura è diventato determinante per lo studio delle macromolecole biologiche. Attraverso l&#39;uso della strumentazione e procedure di metodi angolari specifici e preparazione, i dati sperimentali dei SWAXS visualizza la caratterizzazione atomica e nano-scala di macromolecole.

Piccole e Wide Angle X-Ray Scattering Studi di macromolecole biologiche in soluzione

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 Views.  Rensselaer Polytechnic Institute. La dimostrazione dell'angolo piccolo e grande X-ray scattering (SWAXS) procedura è diventato determinante per lo studio delle macromolecole biologiche. Attraverso l'uso della strumentazione e procedure di metodi angolari specifici e preparazione, i dati sperimentali dei SWAXS visualizza la caratterizzazione atomica e nano-scala di macromolecole.

Video: 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.

Fabbricazione di elettrochimica-DNA biosensori per il rilevamento Reagentless di acidi nucleici, proteine ​​e piccole molecole

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 di assemblee biologiche in liquido

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

This paper presents the methods used for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of Atractosteus spatula (A. spatula) using nanoindentation, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray computed tomography (X-ray CT). The experimental results have been used to investigate the design principles of protective biological materials.

Caratterizzazione di Fish Scales Sovrapposizione (<em&gt; Atractosteus spatola</em&gt;) Usando Nanoindentazione, X-ray CT, FTIR e SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle ...

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.
In this paper, we provide a protocol to investigate samples with characteristic size levels spanning a wide length scale and exhibiting ordering in the mesoscale structure using KWS-2. We present in detail how to use the multiple working modes that are offered by the instrument and the level of performance that is achieved.

Here, we present a protocol to investigate soft matter and biophysical systems over a wide mesoscopic length scale, from nm to &#181;m that involves the use of the KWS-2 SANS diffractometer at high intensities and an adjustable resolution.

Studiare Soft-materia e sistemi biologici per una lunghezza scala larga da nanometri e micrometro Taglie al piccolo angolo Neutron Diffrattometro 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 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.

Il arterovenosa (AV) loop in un modello di Piccoli Animali per studiare Angiogenesi e ingegneria dei tessuti vascolarizzati

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.

Una tecnica innovativa per la generazione e Observing Chemiluminescenza in un ambiente biologico

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.

Patterning bioattivi proteine o peptidi su idrogel utilizzando fotochimica per applicazioni biologiche

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.

Sviluppo di dispositivi microfluidici per studiare la capacità di allungamento di punta-coltivazione di cellule vegetali in spazi estremamente ridotti

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.

Utilizzando Nanoplasmon-diffusione migliorata e imaging del microscopio a basso ingrandimento per quantificare gli esosomi derivati dal tumore

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).

Uso della micrografia computerizzata a raggi X con preparazione dell'acido fosfongstico per visualizzare il tessuto fibromuscolare umano

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