Os autores reconhecem o Dr. Michael J. Friedlander, Diretor do Instituto de Pesquisa Virginia Tech Carilion por incentivar nossos esforços de pesquisa. Este projeto foi apoiado por fundos de desenvolvimento para S.M.M e D.F.K. e, em parte, pela iniciativa Nano-Bio do Institute for Critical Technology and Applied Science da Virginia Tech.

1. Prepare os dispositivos de captura de afinidade16
<ol><li>Limpe o nitreto de silício E-chips incubando-os em 15 ml de acetona por 2 min seguido por 15 ml de metanol por 2 min(Figura 1A). Deixe os chips secarem sob o fluxo de ar laminar.</li>
	<li>Incubar os chips secos em uma placa de agitação aquecida (sem mexer) durante 1,5 h a 150 °C, em seguida, deixe esfriar até a temperatura ambiente antes de usar.</li>
	<li>Use seringas Hamilton para compor misturas lipídicas em pequenos tubos de vidro para conter 25% de clorofórmio, 55% DLPC (1,2-dilauroyl-fosfocolina) em clorofórmio (1 mg/ml) e lipídio ni-NTA de 20% (1,2-dioleoyl-iminodiacético ácido-succinyl-sal) em clorofórmio (1 mg/ml) para um volume total de 40 μl.</li>
	<li>Aplique uma alíquota de 1 μl da mistura sobre cada gota de 15 μl de água Milli-Q em um pedaço de Parafilm em uma placa de Petri úmida(Figura 1B). Incubar amostras no gelo por pelo menos 60 minutos.</li>
</ol>2. Capturar macromoléculas17
<ol><li>Coloque um E-chip com um espaçador integrado de 150 nm em cima de uma amostra de monocamadas e incubar por 1 min(Figura 1C).</li>
	<li>Retire suavemente o chip da amostra e adicione uma alíquota de 3 μl de Proteína A. Incubar por 1 min a temperatura ambiente(Figura 1D).</li>
	<li>Limpe a gota de excesso usando o Whatman #1 papel filtro e adicione imediatamente uma alíquota de 3 μl de solução de anticorpos. Incubar por 1 min a temperatura ambiente.</li>
	<li>Remova a solução em excesso usando uma seringa Hamilton e adicione imediatamente uma alíquota de 1 μl de DLPs rotavírus (0,1 mg/ml) em solução tampão contendo HEPES de 50 mM (pH 7.5), 150 mM NaCl, 10 mM MgCl2 e 10 mM CaCl2. Incubar por pelo menos 2 minutos em temperatura ambiente. A preparação dos DLPs foi descrita anteriormente18.
</li>
</ol>3. Monte a Câmara Microfluiídica e Carregue o Porta-Espécimes In situ
<ol><li>Carregue o e-chip molhado contendo a amostra viral na ponta do suporte da amostra microfluida. Descarregue um segundo e-chip plano por 1 min e depois carregado em cima do chip espaçador(Figura 2, painéis 1-5).</li>
	<li>Alternativamente, para aumentar o contraste das macromoléculas biológicas, a mancha de metal pesado (por exemplo, 0,2% de formação de urano) pode ser adicionada a espécimes molhados antes de colocar o segundo e-chip no espécime molhado. No entanto, o e-chip contendo o espécime precisará ser lavado com água Milli-Q antes de adicionar o reagente de contraste.</li>
	<li>Sanduíche toda a montagem para formar um gabinete selado, mantido no lugar mecanicamente dentro do suporte por 3 parafusos de latão (Figura 2, painéis 6-8).</li>
	<li>Após o montagem, a ponta do suporte é bombeada para 10-6 Torr usando uma estação de bombeamento a seco turbo-bombeada antes de colocar o suporte dentro do TEM.</li>
</ol>4. Imagem em líquido usando um microscópio eletrônico de transmissão
<ol><li>Carregue o porta-espécimes in situ em um microscópio eletrônico de transmissão (FEI Company) equipado com um filamento LaB6 e operando a 120 kV.</li>
	<li>Ligue o filamento TEM e ajuste a altura eucêntrica do estágio do microscópio em relação à amostra usando a função wobbler para inclinar a amostra de -15° para +15° para frente e para trás na coluna. Este procedimento ajusta o estágio na direção z para ajudar a explicar a espessura adequada da câmara líquida. Esta etapa também garante que uma ampliação precisa seja usada ao gravar imagens.</li>
	<li>Registo as imagens ao longo das bordas e nas regiões do canto da câmara microfluidica primeiro. Essas áreas normalmente contêm a solução mais fina. Registo imagens de amostras em condições de baixa dose (1-3 elétrons/Å2) usando uma câmera CCD. Use ampliações nominais de 6.000X - 30.000X.</li>
	<li>Determine o valor adequado do desfoco focando na borda da câmara fluida. Use um valor de -1,5 μm para gravar imagens a 30.000X de ampliação. Se a solução espessa for encontrada ou se o agente de contraste não for usado na preparação da amostra, use valores de desfoco mais elevados na faixa de -2 a -4 μm.</li>
	<li>Para garantir que a solução esteja contida na câmara microfluida durante os experimentos, concentre o feixe de elétrons até que as bolhas sejam formadas no líquido dentro do dispositivo.</li>
</ol>

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U.S.A. 107, 6298-6303 (2010).</li><li>Dubochet, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-electron+microscopy+of+vitrified+specimens.">Cryo-electron microscopy of vitrified specimens.</a> Q. Rev. Biophys. 21, 129-228 (1988).</li><li>Unwin, P. N., Henderson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+structure+determination+by+electron+microscopy+of+unstained+crystalline+specimens.">Molecular structure determination by electron microscopy of unstained crystalline specimens.</a> J. Mol. 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A autora, Madeline J. Dukes, é funcionária da Protochips, Inc.

Em nosso trabalho apresentado, utilizamos a abordagem de captura de afinidade para dLPs rotavírus de tether para uma plataforma microfluida. Isso permitiu imagens in situ de complexos macromoleculares em um microambiente líquido. A abordagem de captura é significativa em relação a outras técnicas de imagem microfluidica, pois localiza espécimes biológicos na janela de imagem para negar grandes problemas difusivos que surgem durante a gravação de imagens em líquido. No entanto, uma das ...

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

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

Erratum: In situ TEM of Biological Assemblies in Liquid

Um objetivo comum de biólogos e engenheiros é entender o funcionamento interno das máquinas moleculares. Os microscópios eletrônicos de transmissão (TEMs) são instrumentos ideais para visualizar esses detalhes intrincados na resolução quase atômica1-2. Para sustentar o sistema de alto vácuo de um TEM, as amostras biológicas são tipicamente incorporadas em pelíneas de gelo vítreo3, açúcares4,sais de metal pesado5, ou alguma combinação de6. Como resultado, imagens de espécimes incorporados podem revelar apenas instantâneos limitados de processos dinâmicos.
As primeiras tentativas de manter espécimes biológicos hidratados em câmaras de líquidos ambientais foram realizadas por Parsons e colegas utilizando estágios diferenciais de bombeamento. Padrões de difração eletrônica de cristais catalase não manchados foram registrados com sucesso em uma resolução de 3 Å em um estado hidratado7-8. Além disso, domínios lipíduos separados em fases poderiam ser examinados em membranas hidratadas de eritrócitos humanos9-10. No entanto, o movimento causado pela difusão do líquido e sua interferência com o feixe de elétrons, resultou em perda severa de resolução e outros experimentos usando espécimes biológicos não foram tentados até recentemente.
Recém-desenvolvidos foram introduzidos portadores de amostras microfluidas que utilizam microchips semicondutores para formar uma câmara ambiental microes em escala. Estes dispositivos podem manter amostras em líquido enquanto estão posicionados em uma coluna TEM11-12. Esse avanço técnico na imagem TEM permitiu que os pesquisadores vissem, pela primeira vez, eventos progressivos no nível molecular13. Referimo-nos a esta nova modalidade como " microscopia molecularin situ", pois os experimentos agora podem ser realizados "dentro" da coluna EM14-15. O objetivo geral deste método é imaginar conjuntos biológicos em líquido, a fim de observar seus comportamentos dinâmicos na resolução de nanômetros. A lógica por trás da técnica desenvolvida é registrar observações em tempo real e examinar novas propriedades do maquinário biológico em solução. Essa metodologia amplia o uso de TEMs para fins mais amplos em biologia celular e molecular12-16.
No artigo de vídeo atual, apresentamos um protocolo abrangente para montar e usar um suporte de espécime microfluidic comercialmente disponível. Esses suportes especializados utilizam microchips de nitreto de silício produzidos com espaçadores integrados para formar uma câmara líquida que inclua volumes minuciosos de solução. Janelas finas e transparentes são gravadas nos microchips para fins de imagem12. Demonstramos o uso adequado de um suporte microfluido para examinar partículas de rotavírus de dupla camada (DLPs) em camadas sídicas em líquido usando um TEM. Para garantir que os conjuntos biológicos, como os DLPs, não se difundam rapidamente em grandes distâncias durante a imagem, empregamos a abordagem Affinity Capture para amarrá-los à superfície da câmara microfluidica16. Esta etapa de captura molecular tem uma grande vantagem sobre técnicas alternativas para imagens de amostras biológicas em líquido, pois permite a aquisição de imagens que serão usadas para rotinas de processamento a jusante. Esta etapa de captura usada em conjunto com a imagem microfluidica é exclusiva dos nossos procedimentos17. Os leitores que utilizam aplicações de biologia estrutural usando câmaras de imagem TEM ou microfluidic podem considerar o uso de técnicas de captura de afinidade quando observações dinâmicas no nível molecular são o objetivo final.

<table border="0" cellpadding="0" cellspacing="0" width="832">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:237px;">E-chips, spacer chip</td>
			<td style="width:183px;">Protochips, Inc.</td>
			<td style="width:245px;">EPB-52TBD</td>
			<td style="width:167px;">400 &#956;m x 50 &#956;m window</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:237px;">E-chip, top chip</td>
			<td style="width:183px;">Protochips, Inc.</td>
			<td style="width:245px;">EPT-45W</td>
			<td style="width:167px;">400 &#956;m x 50 &#956;m window</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Ni-NTA lipid</td>
			<td style="width:183px;">Avanti Polar Lipids</td>
			<td style="width:245px;">790404P</td>
			<td style="width:167px;">Powder form</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">DLPC (12:0) lipid</td>
			<td style="width:183px;">Avanti Polar Lipids</td>
			<td style="width:245px;">850335P</td>
			<td style="width:167px;">Powder form</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Volumetric flasks&#160;</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">20-812A; 20-812C</td>
			<td style="width:167px;">1 ml; 5 ml&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Hamilton Syringes</td>
			<td style="width:183px;">Hamilton Co.</td>
			<td style="width:245px;">80300, 80400</td>
			<td style="width:167px;">1-10 &#956;l; 1-25 &#956;l</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Whatman #1 filter paper</td>
			<td style="width:183px;">Whatman</td>
			<td style="width:245px;">1001 090</td>
			<td style="width:167px;">100 pieces, 90 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glass Petri dishes</td>
			<td style="width:183px;">Corning&#160;</td>
			<td style="width:245px;">70165-101</td>
			<td style="width:167px;">100 mm x 15 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glass Pasteur pipettes</td>
			<td style="width:183px;">VWR</td>
			<td style="width:245px;">14673-010</td>
			<td style="width:167px;">14673-010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glass culture tubes</td>
			<td style="width:183px;">VWR</td>
			<td style="width:245px;">47729-566</td>
			<td style="width:167px;">6 mm x 50 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Acetone</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">&#160;A11-1</td>
			<td style="width:167px;">1 L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Methanol</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">&#160;A412-1</td>
			<td style="width:167px;">1 L</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">Chloroform&#160;</td>
			<td style="width:183px;">Electron Microcopy Sciences</td>
			<td style="width:245px;">12550</td>
			<td style="width:167px;">100 ml&#160;</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">His-tagged Protein A</td>
			<td style="width:183px;">Abcam, Inc.</td>
			<td style="width:245px;">ab52953</td>
			<td style="width:167px;">10 mg</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">Milli-Q water system</td>
			<td style="width:183px;">EMD Millipore Corp.</td>
			<td style="width:245px;">Z00QSV001</td>
			<td style="width:167px;">Ultrapure Water</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">HEPES</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">BP310-500</td>
			<td style="width:167px;">500 g</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">Equipment&#160;</td>
			<td style="width:183px;"></td>
			<td style="width:245px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">Poseidon In situ specimen holder</td>
			<td style="width:183px;">Protochips, Inc.</td>
			<td style="width:245px;"></td>
			<td>&#160;FEI compatible</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">FEI Spirit BioTwin TEM</td>
			<td style="width:183px;">FEI Co.</td>
			<td style="width:245px;"></td>
			<td>120 kV</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">Eagle 2k HS CCD camera</td>
			<td style="width:183px;">FEI Co.</td>
			<td style="width:245px;"></td>
			<td>10&#160;&#197;/pixel sampling at 30,000X</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:237px;">Gatan 655 Dry pump station</td>
			<td style="width:183px;">Gatan, Inc.</td>
			<td style="width:245px;"></td>
			<td>Pump holder tip to 10-6&#160;range</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:237px;">PELCO easiGlow, glow discharge unit</td>
			<td style="width:183px;">Ted Pella, Inc.</td>
			<td style="width:245px;"></td>
			<td>&#160;Negative polarity mode</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Isotemp heated stir plate</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;"></td>
			<td>Heat to 150 &#186;C for 1.5 hr</td>
		</tr>
	</tbody>
</table>

in situ tem of biological assemblies in liquid

Os pesquisadores usam regularmente microscópios eletrônicos de transmissão (TEMs) para examinar entidades biológicas e avaliar novos materiais. Aqui, descrevemos uma aplicação adicional para esses instrumentos: visualização de conjuntos virais em um ambiente líquido. Este excitante e novo método de visualização de estruturas biológicas utiliza um suporte de espécimes de base microfluidic recentemente desenvolvido. Nosso artigo em vídeo demonstra como montar e usar um suporte microfluido para imagem de amostras líquidas dentro de um TEM. Em particular, usamos partículas de rotavírus de dupla camada (DLPs) como nosso sistema modelo. Também descrevemos passos para revestir a superfície da câmara líquida com biofilmes de afinidade que amarram DLPs à janela de visualização. Isso nos permite fazer assembleias de imagem de forma adequada para a determinação da estrutura 3D. Assim, apresentamos um primeiro vislumbre de partículas subvirais em um ambiente líquido nativo.

Imagens representativas de DLPs em líquido usando E-chips que foram descarregadas por brilho(Figura 3A) mostram menos DLPs em uma determinada área de visualização, presumivelmente devido à difusão, em comparação com DLPs que são enriquecidos em dispositivos affinity capture(Figura 3B). A adição de formato de urano na câmara de imagem aumenta o contraste da amostra e, consequentemente, a visibilidade de DLPs individuais na solução(Figura 3C, painel superior)...

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

In situ TEM of Biological Assemblies in Liquid

Aqui descrevemos um procedimento para imagem de complexos virais em liquidez em resolução de nanômetros usando um microscópio eletrônico de transmissão.

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 ...

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

Research

JoVE Journal

Bioengineering

In situ TEM of Biological Assemblies in Liquid

9.9K visualizações.  Protochips, Inc.. Aqui descrevemos um procedimento para imagem de complexos virais em liquidez em resolução de nanômetros usando um microscópio eletrônico de transmissão.

Vídeo: In situ TEM of Biological Assemblies in Liquid

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

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

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

Extração de lipídeos celulares para Targeted diluição de isótopos estáveis ​​Líquido Análise Espectrometria de Massa-Cromatografia

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

Bioengenharia

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

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

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

Quantificação da função global diastólica por análise baseada em Modelagem cinemática de transmitral fluxo através do formalismo de enchimento parametrizada diastólica

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

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

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

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

Geração de escalável e de alto-Aspect Metallic nanocompósitos índices, em um Biológico Líquido Médio

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

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

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

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

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

Em Situ Mapeamento das propriedades mecânicas de biofilmes por de rastreamento de partículas Microrheology

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

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

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

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

Alternando microgéis de gelatina híbrido Campo-Responsive magnética para controlada de fármacos de libertação

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

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

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

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

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 ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

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

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

Robótico líquido manipulação montagem de dispositivos modulares DNA automática

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

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

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

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

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

A<em&gt; In Vitro</em&gt; Modelo Cultura Órgão da Murino Intervertebral Disc

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

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

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

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

Síntese de biocompatíveis cristal líquido de elastómero Espumas como andaimes celulares para cultura de células 3D espaciais

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

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

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

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

Dispositivos de campo elétrico e magnético para estimulação de tecidos biológicos

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

Methods Article

In situ TEM de Assembleias Biológicas em Líquido