Gostaríamos de agradecer ao Dr. Manfred Kriechbaum de XRS Hecus e do Instituto de Biofísica e Pesquisa Nanosystems da Academia Austríaca de Ciências, em Graz, na Áustria. LL e XW foram apoiados em parte pelo Departamento de Energia dos EUA, sob NERI-C Prêmio N ° DE-FG07-07ID14889, e dos EUA Comissão Reguladora Nuclear, sob n º Prêmio NRC-38-08-950. O instrumento SWAXS também é suportado, em parte, pelo Departamento de Energia dos EUA, sob Prêmio N ° DE-NE0000325.

1. Preparação da Amostra <ol><li> Use uma agulha para retirar alguma da amostra a partir do recipiente da amostra. * </li><li> Usar a agulha para encher o capilar (diâmetro máximo de 2,2 mm) com a amostra. O capilar deve ser cheio entre 2 e 3 cm do fundo. </li><li> Feche o capilar por cera derretida em sua ponta. </li><li> Desaparafusar o suporte de amostra do sistema de vácuo. </li><li> Leve o capilar até o final fundido (fim de cera) e insira a ponta não-fundido no suporte da amostra. </li><li> Colocar o suporte de volta para o sistema e o parafuso no lugar. </li></ol> * As amostras sólidas (incluindo as amostras de pó) pode ser directamente colocada no suporte da amostra (sem necessidade de capilar), ao passo que as amostras de líquido deve ser colocado em um tubo capilar. Start-Up da Máquina SWAXS 2. Fonte procedimento de inicialização a frio <ol><li> Feche o obturador, pressionando o botão do obturador de segurança. </li><li> Mudar a alimentação principal&quot;ON&quot;, empurrando o verde no botão. </li><li> Verifique se o Emergency &quot;Shut-Off&quot; está na posição estendida. </li><li> Mudar a alimentação principal, pressionando o botão verde. </li><li> Verifique se o bloqueio de luz LED fica verde, indicando todos os bloqueios são ok. </li><li> Aguarde a tela de toque para carregar. Uma vez carregado, pressione &quot;R6&quot; no menu. </li><li> Gire a chave de espera para a posição &quot;ON&quot;. </li><li> Gire a chave de raios-X para a posição &quot;ON&quot;. A luz vermelha de raios-X deve acender. </li><li> Aguarde a tela de toque para ler os níveis de reserva (30 kV e 0,4 mA). </li><li> Gire a chave de segurança de espera para a posição &quot;OFF&quot;. Pressione o botão &quot;Iniciar Ciclo&quot; botão na parte inferior da tela sensível ao toque. </li><li> Aguarde a tela de toque para ler potência nominal (50 kV e 1 mA). Se um nível de potência diferente for desejada, entrar na tela de configuração pressionando &quot;R4&quot; na tela de toque e digitar as configurações desejadas. </li></ol> 3. Procedimento Chiller <ol><li> Vire o swi podertch para a posição &quot;ON&quot; no painel de controle de temperatura. A luz de controle eo indicador de temperatura LED acenderá. </li><li> Ligue o interruptor para a posição &quot;ON&quot; no chiller. </li><li> Aguarde até que o indicador de temperatura lê &quot;OFF&quot;. Este é o modo de espera. </li><li> Pressione e segure o botão enter (símbolo de retorno) por cerca de 4 segundos ou até que o indicador de temperatura indica uma temperatura real. </li><li> Para alterar a temperatura, pressione para cima ou seta para baixo. O valor ajustado será apresentado no visor de temperatura para cerca de 8 segundos antes de regressar ao valor real. </li><li> Pressione o botão Enter uma vez que o valor conjunto desejado seja exibido. Isto vai armazenar esse valor. </li><li> Aguarde até que a temperatura real atinge o valor ajustado desejado. </li></ol> NOTA: Se a qualquer momento o erro &quot;E 01&quot; aparece no visor da temperatura, siga as instruções Chiller desligamento, recarga da unidade banho de água purificada por vazamento no tanque de fill, e em seguida, executar o procedimento de inicialização chiller novamente. 4. Desligamento de Chiller <ol><li> Pressione &quot;Enter&quot; para cerca de 4 seg. </li><li> Ligue o interruptor para a posição &quot;OFF&quot;. </li></ol> 5. Ligar o vácuo <ol><li> Garantir a porta de vácuo é mantido em seu lugar. </li><li> Vire o vácuo 1 e 2 botões para o &quot;on&quot; posições. </li><li> Espere até que o manómetro de vácuo VAP5 lê um nível de vácuo inferior a 1,5 mbar. </li></ol> 6. Configuração do Sistema detector <ol><li> Para configurar a pressão do gás, verifique se o manômetro principal da válvula de redução de pelo menos 10 bar. </li><li> Abrir a válvula principal. </li><li> Abra lentamente a válvula de pressão de operação até que a pressão atinge 8 bar no manômetro operacional. </li><li> Abra a segunda válvula principal </li><li> Para ajustar o fluxo de gás, abra a válvula de pressão principal, girando o botão para a posição vertical no painel de controle de gás. </li><li> Ajustar o fluxotaxa com a válvula de agulha até que o medidor lê a cerca de 8 bar. </li><li> Ligue o interruptor principal para a posição &quot;ON&quot;. O LED deve acender. </li><li> Para ajustar a tensão alta, ativar a alta tensão, girando o botão para a posição &quot;ON&quot;. O LED deve acender. </li><li> Lentamente gire o botão de controle de tensão até aproximadamente 3.5kV é atingido no medidor. NÃO ULTRAPASSAR 4kV. </li></ol> 7. Calibragem <ol><li> Os passos experimentais acima são realizados com uma amostra de picos conhecidos. </li><li> ASA 3.3 é usado para obter o gráfico da intensidade vs canal com 5 picos. </li><li> Vá para Menu → Opções → Digite canais e intensidades correspondentes. </li><li> Ir para ASA3, &quot;TPF&quot; guia primeiro. </li><li> Mudar o filtro, parte superior esquerda um milímetro de níquel (feixe de filtro). </li><li> Definir a posição 32.000 (faixa de 28.000 a 32.000, região crítica em torno de 28.000) → ir para a posição, fazer vácuo certeza está abaixo de 5 mbar, certifique-se que o detector é reading entre 1k-10K. </li><li> Correr durante 10 segundos para cerca de 52 contagens por segundo. </li><li> Alterar as janelas de energia, (ou medir a janela de energia com a amostra apenas, não incluindo o caso do filtro). Definir a corrida final por 10 seg. </li><li> Encontrar a localização do feixe primário (233 de canal, por exemplo). </li><li> Para outras corridas, mudar filtrar novamente a 2 milímetros de tungstênio (W, feixe de rolha) e começar a partir de 30.000. </li><li> Encontrar a localização de todos os outros picos das amostras. </li><li> Use ImageJ macro-software para calibrar SAXS - fazer transições de intensidade versus canal de intensidade versus q (espaçamento d ou) (Figura 1). </li><li> Use ImageJ macro-software para calibrar WAXS - fazer transições de intensidade versus canal de intensidade versus q (espaçamento d ou) (Figura 2). </li></ol> 8. Procedimento software <ol><li> Use o software ASA 3.3, para coletar os dados ao vivo: Live Data → TPF guia → Salvar arquivo. </li><li> Para EasySWAXS, clique na guia Configurações do dispositivo. </li><li> Introduzir o &quot;a&quot;, lambda, e os valores de d, assim como o centro de gravidade. </li><li> Selecione Colimação Point. </li><li> Seleccione o tipo de partículas globulares (a menos que seja um conhecido por ser uma haste de partícula em forma plana). </li><li> Clique na aba Guinier-Plot. Um eu vs q 2 gráfico será exibido. </li><li> Arraste as linhas verticais para cercar uma seção de inclinação aproximadamente linear. </li><li> Na parte inferior da tela, um valor de R será exibida. Em seguida a isso, há uma exigência de validação. </li><li> Continue a arrastar as linhas verticais mais juntos até que o valor de validação está entre 1 e 2. Isso é subjetivo. O valor de R, ou raio de giro, obtido é uma estimativa. </li></ol> 9. Procedimento Shutdown fonte <ol><li> Verificar a segurança e persianas rápidas estão fechados. </li><li> Gire a chave de espera para a posição &quot;ON&quot;. </li><li> Aguarde até que o ecrã táctil para ler níveis de espera. </li><li> Imprensa R5 no touchscreen. </li><li> Reduzir a corrente a 0 A. </li><li> Reduzir a tensão a 0 A. </li><li> Gire a chave de raios-X para a posição &quot;OFF&quot;. </li><li> Mude o principal poder &quot;OFF&quot; pressionando o botão de desligar vermelho. </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>

Não há conflitos de interesse declarados.

O procedimento comparativo do sistema SWAXS permite numerosas variáveis ​​a serem determinadas a partir da análise experimental. Os parâmetros que são obtidos a partir da análise podem ser usados ​​para diferentes finalidades de acordo com a amostra e montagem experimental. SAXS fornece informações sobre o nano-escala de tamanho e forma do objecto, enquanto que se concentra em WAXS estrutura atómica e micro-escala (eg molecular reticulado, unidade de simetria dimensão da célula). Mais especifi...

<table border="1"><tbody><tr><td> Nome dos produtos </td><td> Companhia </td></tr><tr><td> O System3 pequena e grande-angular de espalhamento de raios X (SWAXS) Camera </td><td> Hecus XRS e IBN, Graz, Áustria </td></tr><tr><td> GNOM </td><td> ATSAS pacote de 2,3 por D. Svergun EMBL-Hamburgo </td></tr></tbody></table>

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

Neste artigo, Pequenas e Wide Angle X-ray Scattering análise (SWAXS) de macromoléculas é demonstrado através da experimentação. SWAXS é uma técnica na qual os raios X são elasticamente espalhados por uma amostra não homogéneo na gama nm-em ângulos pequenos (tipicamente 0,1 - 5 °) e ângulos de largura (normalmente&gt; 5 °). Esta técnica fornece informações sobre a forma, tamanho e distribuição de macromoléculas, distâncias característicos de materiais parcialmente ordenados, tamanhos de poro, e de superfície-para-volume. Pequeno ângulo de raios X Scattering (SAXS) é capaz de fornecer informação estrutural de macromoléculas entre 1 e 200 nm, enquanto Wide Angle X-ray Scattering (WAXS) podem resolver ainda menor espaçamento Bragg de amostras entre 0,33 nm e 0,49 nm com base na configuração do sistema específico e detector. O espaçamento é determinada a partir da lei de Bragg e é dependente do comprimento de onda e do ângulo de incidência. Em uma experiência SWAXS, os materiais podem ser sólidasou um líquido e pode conter sólidos, líquidos ou gasosos domínios (os chamados partículas) do mesmo material ou de outro, em qualquer combinação. Aplicações SWAXS são muito amplos e incluem colóides de todos os tipos: metais, compósitos, cimento, petróleo, polímeros, plásticos, proteínas, alimentos e produtos farmacêuticos. Para amostras sólidas, a espessura é limitado a cerca de 5 mm. O uso de um instrumento SWAXS baseado em laboratório é detalhado neste documento. Com o software disponível (por exemplo, GNOM-ATSAS pacote 2,3 por D. Svergun EMBL-Hamburg e software EasySWAXS) para o sistema SWAXS, uma experiência pode ser conduzida para determinar certos parâmetros de interesse para a amostra dada. Um exemplo de uma experiência de macromolécula biológica é a análise de 2% em peso de lisozima num tampão à base de água aquosa que pode ser escolhido e preparado através de vários métodos. A preparação da amostra segue as orientações abaixo na preparação da seção da amostra. Através da experimentação SWAXS,importantes parâmetros estruturais da lisozima, por exemplo, o raio de rotação, podem ser analisadas.

SAXS e WAXS completo pode fornecer informações sobre a estrutura da amostra através dos seguintes parâmetros: o raio de giração de tamanho de partícula e forma, o factor de estrutura de solução, superfície interna específica e dimensão de poro, tipo treliça e dimensão e densidade electrónica 1. SAXS e WAXS pode também ser aplicado ao estudo da dinâmica das proteínas 2. A informação estrutural de experiências SWAXS é obtida através da comparação ...

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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

A demonstração do ângulo pequeno e amplo dispersão de raios X procedimento (SWAXS) tornou-se essencial para o estudo de macromoléculas biológicas. Através da utilização da instrumentação e procedimentos de métodos específicos de ângulo e de preparação, os dados experimentais dos SWAXS mostra a caracterização atómica e nano-escala de macromoléculas.

Pequenas e Wide Angle X-Ray Scattering Estudos de macromoléculas biológicas em solução

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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18.0K visualizações.  Rensselaer Polytechnic Institute. A demonstração do ângulo pequeno e amplo dispersão de raios X procedimento (SWAXS) tornou-se essencial para o estudo de macromoléculas biológicas. Através da utilização da instrumentação e procedimentos de métodos específicos de ângulo e de preparação, os dados experimentais dos SWAXS mostra a caracterização atómica e nano-escala de macromoléculas.

Vídeo: 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.

Fabricação de Eletroquímica-DNA Biossensores para Detecção Reagentless de ácidos nucléicos, proteínas e pequenas moléculas

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. ...

Bioengenharia

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 Assembleias Biológicas em 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.

Caracterização de escamas de peixe multi-camadas (<em&gt; Atractosteus espátula</em&gt;) Usando Nanoindentação, 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.

Estudar Soft-matéria e Sistemas Biológicos longo de um comprimento escala larga de nanômetros e micrômetro Tamanhos no pequeno ângulo Neutron Difractï¿½etro 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.

The Loop arteriovenosa (AV) em um modelo animal pequeno para estudar angiogénese e Engenharia de tecido 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.

Uma nova técnica para gerar e Observing Quimioluminescência em um ambiente Biológica

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.

Padronização bioativas proteínas ou peptídeos em hidrogel usando fotoquímica para aplicações biológicas

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.

Desenvolvimento de dispositivos microfluídicos para estudar a capacidade de alongamento de ponta-cultivo de células vegetais em espaços extremamente pequenos

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 Nanoplasmon-Enhanced espalhamento e microscópio de baixa ampliação de imagem para quantificar Exosomas derivados do 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 do micro tomography computado do raio X com preparação ácida phosphotungstic para visualizar o tecido fibromuscular humano

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