Nos gustaría dar las gracias al Dr. Manfred Kriechbaum de XRS Hecus y el Instituto de Biofísica y nanosistemas Investigación de la Academia Austriaca de Ciencias en Graz, Austria. LL y XW se apoya en parte por EE.UU. Departamento de Energía, bajo NERI-C Laudo No. DE-FG07-07ID14889 y los EE.UU. Comisión de Regulación Nuclear, bajo la Donación No. NRC-38-08-950. El instrumento SWAXS también es apoyado en parte por el Departamento de Energía de EE.UU., bajo la Donación No. DE-NE0000325.

1. Preparación de la muestra <ol><li> Utilice una aguja para extraer parte de la muestra desde el recipiente de la muestra. * </li><li> Utilice la aguja para llenar el capilar (diámetro máximo de 2,2 mm) con muestra. El capilar debe ser llenado entre 2 y 3 cm de la parte inferior. </li><li> Cierre el capilar por cera derretida en la punta. </li><li> Desatornillar el soporte de muestra de vacío del sistema. </li><li> Tome el capilar por el extremo fundido (extremo cera) e inserte el extremo no-condensado en el soporte de la muestra. </li><li> Coloque el soporte de nuevo en el sistema y el tornillo en su lugar. </li></ol> * Las muestras sólidas (incluyendo muestras de polvo) puede ser colocado directamente en el soporte de muestras (no capilar es necesario), mientras que las muestras de líquido debe ser colocado en un capilar. Puesta en marcha de la máquina SWAXS 2. Fuente Procedimiento de arranque en frío <ol><li> Cierre el obturador pulsando el botón de disparo hasta la seguridad. </li><li> Encienda la alimentación principal&quot;ON&quot; pulsando el botón verde en. </li><li> Verifique que la emergencia &quot;Shut-Off&quot; está en la posición extendida. </li><li> Encienda la alimentación principal pulsando el botón verde. </li><li> Verifique que la luz LED de bloqueo es de color verde, indicando todos los bloqueos están bien. </li><li> Espere a que la pantalla táctil para cargar. Una vez que se haya cargado, pulse &quot;R6&quot; en el menú. </li><li> Gire la llave de espera para la posición &quot;ON&quot;. </li><li> Gire la Llave de rayos X a la posición &quot;ON&quot;. El rojo de luz de rayos X debe encenderse. </li><li> Espere a que la pantalla táctil para leer los niveles de reserva (30 kV y 0,4 mA). </li><li> Gire la llave de seguridad en espera a la posición &quot;OFF&quot;. Pulse la tecla &quot;Cycle Start&quot; en la parte inferior de la pantalla táctil. </li><li> Espere a que la pantalla táctil para leer potencia nominal (50 kV y mA 1). Si el nivel de potencia diferente que se desea, entrar en la pantalla de configuración pulsando &quot;R4&quot; en la pantalla táctil e introducir los ajustes deseados. </li></ol> 3. Chiller Procedimiento <ol><li> Gire el swi podertch a la posición &quot;ON&quot; en el panel de control de temperatura. Una luz de control y la pantalla de temperatura LED se iluminará. </li><li> Gire el interruptor de encendido en la posición &quot;ON&quot; en la enfriadora. </li><li> Espere hasta que el indicador de temperatura muestra &quot;OFF&quot;. Este es el modo de espera. </li><li> Mantenga pulsado el botón de entrar (símbolo de retorno) durante unos 4 segundos o hasta que el indicador de temperatura muestra la temperatura actual. </li><li> Para cambiar la temperatura, presione las flechas arriba hacia abajo. El valor de ajuste se muestra en la pantalla de temperatura durante aproximadamente 8 segundos hasta que se vuelva al valor real. </li><li> Pulse el botón Enter una vez el valor ajustado deseado. Esto almacenará ese valor. </li><li> Esperar hasta que la temperatura real alcanza el valor de referencia deseado. </li></ol> NOTA: Si en cualquier momento el error &quot;E 01&quot; aparece en la pantalla de temperatura, siga las instrucciones de apagado refrigerador, baño de recarga de la unidad mediante el vertido de agua purificada en el filtro del tanquel, a continuación, realice el procedimiento de inicio de enfriadores de nuevo. 4. Parada de Chiller <ol><li> Pulse &quot;enter&quot; durante aproximadamente 4 segundos. </li><li> Gire el interruptor de encendido en la posición &quot;OFF&quot;. </li></ol> 5. Encendido del vacío <ol><li> Asegúrese de que la puerta de vacío se fija en su lugar. </li><li> Encienda la aspiradora 1 y 2 a los mandos de posiciones &quot;ON&quot;. </li><li> Espere hasta que el indicador de vacío VAP5 lee un nivel de vacío inferior a 1,5 mbar. </li></ol> 6. Detector de configuración del sistema <ol><li> Para configurar la presión del gas, verifique que el manómetro principal de la válvula de reducción es de al menos 10 bar. </li><li> Abra la válvula principal. </li><li> Abrir lentamente la válvula de presión de funcionamiento hasta que la presión alcanza 8 bar en el medidor de presión de trabajo. </li><li> Abra la válvula principal Segundo </li><li> Para ajustar el flujo de gas, abra la válvula de presión principal girando la perilla a la posición vertical en el panel de control de gas. </li><li> Ajuste el flujo develocidad con la válvula de aguja hasta que el manómetro marque aproximadamente 8 bar. </li><li> Gire el interruptor de alimentación principal a la posición &quot;ON&quot;. La luz LED se iluminará. </li><li> Para ajustar la tensión alta, activar la alta tensión girando el interruptor en la posición &quot;ON&quot;. La luz LED se iluminará. </li><li> Gire lentamente la perilla de control de voltaje de hasta aproximadamente 3.5kV se alcanza en el manómetro. NO EXCEDA 4kV. </li></ol> 7. Calibración <ol><li> Las etapas experimentales anteriores se realizan con una muestra de picos conocidos. </li><li> ASA 3,3 se utiliza para obtener el gráfico de intensidad vs canal con 5 picos. </li><li> Vaya a Menú → Opciones → Introducir canales y las intensidades correspondientes. </li><li> Ir a ASA3 &quot;, TPF&quot; primera ficha. </li><li> Cambiar el filtro, arriba a la izquierda 1 mm Níquel (haz filtro). </li><li> Establece la posición 32.000 (rango 28.000 a 32.000, región crítica en torno a 28.000) → pasar a la posición, hacer vacío que es inferior a 5 mbar, asegúrese de que el detector se reading entre 1k-10K. </li><li> Corre por 10 segundos durante aproximadamente 52 cuentas por segundo. </li><li> Cambiar las ventanas de energía, (o medir la ventana de energía con la muestra solamente, sin incluir la caja del filtro). Ajuste la recta final durante 10 segundos. </li><li> Encontrar la ubicación de la viga principal (233 canal, por ejemplo). </li><li> Para otras carreras, cambie el filtro de nuevo en Tungsteno mm 2 (W, haz tapón) y empezar de 30.000. </li><li> Encuentre la ubicación de todos los otros picos de las muestras. </li><li> Use ImageJ-macro software para calibrar SAXS - hacer transiciones de intensidad vs canal de intensidad vs q (o espaciado d) (Figura 1). </li><li> Use ImageJ-macro software para calibrar WAXS - hacer transiciones de intensidad vs canal de intensidad vs q (o espaciado d) (Figura 2). </li></ol> 8. Software Procedimiento <ol><li> Utilice el software ASA 3.3, para recoger los datos en vivo: Live Data TPF → → ficha Guardar archivo. </li><li> Para EasySWAXS, haga clic en la ficha Configuración de dispositivo. </li><li> Introduzca la &quot;a&quot;, lambda, y valores d, así como el centro de gravedad. </li><li> Seleccione Colimación Point. </li><li> Seleccionar el tipo de partícula globular (a menos que sea un conocido por ser una varilla de partícula conformada plana). </li><li> Haga clic en la ficha Guinier-Plot. Un I vs q 2 gráfico será mostrado. </li><li> Arrastre las líneas verticales que rodean una sección de pendiente aproximadamente lineal. </li><li> En la parte inferior de la pantalla, un valor de R se mostrará. Junto a esta hay un requisito de validación. </li><li> Continúe arrastrando las líneas verticales más juntos hasta que el valor de validación es entre 1 y 2. Esto es subjetivo. El valor R, o radio de giro, que se obtiene es una estimación. </li></ol> 9. Fuente Procedimiento de apagado <ol><li> Verifique la seguridad y persianas rápidas están cerrados. </li><li> Gire la llave de espera para la posición &quot;ON&quot;. </li><li> Espere a que la pantalla táctil para leer los niveles de reserva. </li><li> Pulse R5 en el touchscreen. </li><li> Reducir la corriente a 0 A. </li><li> Reducir la tensión a 0 A. </li><li> Gire la llave de rayos X a la posición &quot;OFF&quot;. </li><li> Encienda el interruptor principal en &quot;OFF&quot; presionando el botón de color rojo. </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>

No hay conflictos de interés declarado.

El procedimiento comparativo del sistema SWAXS permite numerosas variables que deben determinarse a partir del análisis experimental. Los parámetros que se alcanzan a partir del análisis puede ser usada para fines diferentes de acuerdo con la muestra y la configuración experimental. SAXS proporciona información acerca de nano-escala de tamaño y la forma del objeto, mientras que WAXS se centra en la estructura atómica y micro escala-(por ejemplo, red molecular, celda unidad simetría dimensión). Más esp...

<table border="1"><tbody><tr><td> Nombre de los productos </td><td> Empresa </td></tr><tr><td> El System3 la Pequeña y Gran Angular-X-Ray Scattering (SWAXS) Cámara </td><td> Hecus XRS y IBN, Graz, Austria </td></tr><tr><td> GNOM </td><td> ATSAS 2,3 paquete por D. Svergun EMBL-Hamburg </td></tr></tbody></table>

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

En este trabajo, Ángulo Pequeño y Gran dispersión de rayos X (SWAXS) el análisis de las macromoléculas se demuestra a través de la experimentación. SWAXS es ​​una técnica en la que los rayos X son elásticamente dispersada por una muestra homogénea en el rango de nm-en ángulos pequeños (típicamente 0,1 - 5 °) y ángulos anchos (típicamente&gt; 5 °). Esta técnica proporciona información sobre la forma, el tamaño y la distribución de las macromoléculas, distancias características de materiales parcialmente ordenados, tamaños de poros, y la relación de superficie a volumen. De ángulo pequeño dispersión de rayos X (SAXS) es capaz de entregar información estructural de macromoléculas entre 1 y 200 nm, mientras que Wide Angle X-ray (WAXS) puede resolver aún más pequeño espaciamiento de Bragg de muestras entre 0,33 nm y 0,49 nm basado en la configuración específica del sistema y el detector. La separación se determina a partir de la ley de Bragg y es dependiente de la longitud de onda y ángulo de incidencia. En un experimento SWAXS, los materiales pueden ser sólidoso líquido, y puede contener dominios de sólidos, líquidos o gaseosos (denominadas partículas) del mismo material o de otro en cualquier combinación. SWAXS aplicaciones son muy amplias e incluyen coloides de todo tipo: metales, materiales compuestos, cemento, petróleo, polímeros, plásticos, proteínas, alimentos y productos farmacéuticos. Para muestras sólidas, el espesor está limitado a aproximadamente 5 mm. El uso de un instrumento de laboratorio basado en SWAXS se detalla en este documento. Con el software disponible (por ejemplo, GNOM-ATSAS 2,3 paquete por D. Svergun EMBL-Hamburgo y software EasySWAXS) para el sistema SWAXS, un experimento puede llevarse a cabo para determinar algunos parámetros de interés para la muestra dada. Un ejemplo de un experimento de macromolécula biológica es el análisis de 2% en peso de lisozima en un tampón acuoso a base de agua que puede ser elegido y preparado a través de numerosos métodos. La preparación de la muestra sigue las directrices indicadas a continuación en la Preparación de la sección de muestra. A través de la experimentación SWAXS,importantes parámetros estructurales de la lisozima, por ejemplo, el radio de giro, se puede analizar.

SAXS y WAXS completo puede proporcionar información estructural de la muestra a través de los siguientes parámetros: el radio de giro de tamaño de partícula, y la forma, el factor de estructura de la solución, la superficie interior específica y tamaño de poro, tipo celosía y dimensión, y la densidad de electrones 1. SAXS y WAXS también puede ser aplicado al estudio de la dinámica de las proteínas 2. La información estructural de experimentos SWAXS se obti...

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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 demostración de rayos X de pequeño ángulo y gran dispersión (SWAXS) procedimiento ha resultado fundamental en el estudio de macromoléculas biológicas. Mediante el uso de la instrumentación y los procedimientos de los métodos de ángulo específico y la preparación, los datos experimentales de los SWAXS muestra la caracterización atómica y nano escala de macromoléculas.

Pequeñas y Wide Angle X-Ray Estudios de dispersión de macromoléculas biológicas en disolución

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 vistas.  Rensselaer Polytechnic Institute. La demostración de rayos X de pequeño ángulo y gran dispersión (SWAXS) procedimiento ha resultado fundamental en el estudio de macromoléculas biológicas. Mediante el uso de la instrumentación y los procedimientos de los métodos de ángulo específico y la preparación, los datos experimentales de los SWAXS muestra la caracterización atómica y nano escala de macromoléculas.

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.

Fabricación de ADN electroquímico biosensores para la detección Reagentless de ácidos nucleicos, proteínas y moléculas pequeñas

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 de Ensamblajes Biológicos en Líquido

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.

Caracterización de las escalas de peces de múltiples capas-(<em&gt; Atractosteus espátula</em&gt;) Usando Nanoindentación, X-ray CT, FTIR y 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.

El estudio de la materia y Soft-Sistemas Biológicos en un amplio Longitud escala del nanómetro y el micrómetro Tamaños en el pequeño ángulo de neutrones difractómetro 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.

El asa arteriovenosa (AV) en un modelo de pequeños animales para estudiar la angiogénesis e Ingeniería tejido vascularizado

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 nueva técnica para generar y Observación de quimioluminiscencia en un Contexto biológico

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.

Patrones bioactivas proteínas o péptidos en hidrogel con fotoquímica para usos biológicos

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.

Desarrollo de dispositivos microfluídicos para estudiar la capacidad de elongación de la punta de crecimiento de las células vegetales en espacios extremadamente pequeños

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.

Usando dispersión mejorada por Nanoplasmon y imágenes de microscopio de baja magnificación para cuantificar exosomas derivados del tumor

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 de tomografía computarizada de micro rayos X con preparación de ácido fosfotúrtico para visualizar el tejido fibromuscular humano

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