Este trabalho foi financiado pela National Science Foundation [mecânica de materiais e estruturas programa, atribuir número de 1562656]; e a sociedade americana de engenheiros mecânicos [Haythornthwaite Young Investigator Award].

<ol>
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Os autores não têm nada para divulgar.

Diversas etapas do protocolo são particularmente importantes para garantir que as forças e deslocamentos são medidos com precisão. Enquanto alguns destes passos críticos são universais para todos os testes de flexão de três pontos, outras são exclusivas para este dispositivo de teste mecânico.
Na etapa 1.2 do protocolo o espelho LP é limpo e inspecionado por arranhões e passo 1.6 do protocolo encontra-se o ganho FODS. É importante para o ganho e a...

Ao estudar as arquiteturas de estruturas biológicas (LBBSs), tais como shell e osso de rolamento da carga, os engenheiros desenvolveram novos materiais compósitos que são forte e duro 1. Ficou demonstrado que as propriedades mecânicas notáveis de LBBSs e os seus homólogos de bio-inspirado estão relacionadas com suas arquiteturas internas intricadas 2. No entanto, as relações entre arquiteturas LBBS e propriedades mecânicas não são totalmente compreendidas. Resposta mecânica de um LBBS de medição é o primeiro passo para entender como sua arquitetura melhora suas propriedades mecânicas.
No entanto, é importante que o tipo de teste usado para medir a resposta mecânica de um LBBS é consistente com a sua função mecânica. Por exemplo, uma vez que as penas devem suportar cargas aerodinâmicas, a principal função de um ráquis pena é fornecer a rigidez à flexão 3. Portanto, um teste de dobrando é preferido a um teste de tensão uniaxial para medir sua resposta mecânica. Na verdade, muitos LBBSs — tais como a pena rachises 3, grama caules 4e espículas 5,6,7,8— principalmente se deforma, dobrando. Isto é porque estes LBBSs são delgadas —ou seja, seu comprimento é muito maior que sua largura ou profundidade. No entanto, realizando testes de flexão nestas LBBSs é um desafio porque as forças e deslocamentos que eles podem suportar antes de falhar variam de 10-2 -102 N e 10-4 a 10-3 m, respectivamente 3 , 4 , 5 , 7 , 8. por conseguinte, o dispositivo usado para realizar esses ensaios mecânicos deve ter força e deslocamento resoluções de ≈10-5 N e ≈10-7 m (ou seja, 0,1% da força máxima mensurável e deslocamento do sensor), respectivamente.
Comercialmente disponível, em grande escala, sistemas de ensaios mecânicos normalmente não se pode medir forças e deslocamentos com esta resolução. Enquanto força atômica baseada em microscópio 9,10 ou Microeletromecânicos baseadas em sistemas 11 dispositivos de teste têm resolução adequada, a força máxima (deslocamento respectiva) podem medir é menor do que o força máxima (deslocamento respectivo) que pode suportar a LBBS. Portanto realizar testes de flexão esses LBBSs, engenheiros e cientistas deve contar com Custom-Built mecânico teste dispositivos 5,7,12,13. A principal vantagem destes dispositivos personalizados é que podem acomodar grandes gamas de forças e deslocamentos. No entanto, a construção e o funcionamento destes dispositivos não está bem documentada na literatura.
Um protocolo é descrito, realizando testes de flexão de três pontos usando um dispositivo de teste mecânico personalizado que pode medir as forças que variam de 10-5 a 101 N e deslocamentos variando de 10-7 a 10-2 m. Desenhos técnicos, incluindo todas as dimensões, os componentes do dispositivo de teste mecânico são fornecidos no Material complementar. A principal vantagem deste dispositivo de testes mecânicos é que os intervalos de força e o deslocamento podem ser facilmente ajustados para se adequar a diferentes LBBSs. Princípio de funcionamento do dispositivo é semelhante de um microscópio de força atômica 9. Neste dispositivo, um espécime é colocado através de uma trincheira cortar em uma placa de aço inoxidável (veja a Figura 1A-C). A extensão da trincheira é medida de micrografias ópticas para ser 1278 ± 3 µm (média ± desvio-padrão; n = 10). As bordas de trincheira apoiar a amostra durante um teste de flexão (ver Figura 1e D). Nesta fase de amostra é anexada a um estágio de tradução de três eixos e posicionada debaixo de uma cunha de alumínio para que a cunha está localizada no meio do caminho em toda a extensão da trincheira (ver Figura 1C). Movendo o palco <img alt="Equation 1" src="/files/ftp_upload/56571/56571eq1.jpg"> direção (ver figura 1Ae C), a amostra é empurrada para a cunha causando o espécime para dobrar.
Nos referimos a cunha como a ponta do ponto de carga (LPT) e o componente do dispositivo que contém a cunha como o ponto de carga (LP). O LP é anexado ao fim de um cantilever cujo deslocamento é medido por um sensor de deslocamento óptica de fibra (FODS). O FODS emite luz infravermelha, que é refletida em um espelho localizado na superfície superior do LP (ver Figura 1B) e recebido por uma fibra óptica no FODS. Uma peça quadrada de uma bolacha de silicone polido ≈5 mm é usada como o espelho de LP e aposição do LP usando epóxi. O FODS mede deslocamentos, comparando a intensidade da luz emitida e refletida. A rigidez do cantilever e deslocamento são usados para calcular a força, <img alt="Equation 2" src="/files/ftp_upload/56571/56571eq2.jpg"> </img>, vividas por Cunha devido à sua interação com a amostra. O deslocamento do cantilever também é usado para calcular o deslocamento de seção transversal do espécime sob a cunha, <img alt="Equation 3" src="/files/ftp_upload/56571/56571eq3.jpg"> </img>. Sensores de força baseados em cantilever têm sido usados em um número de micro e macroescala mecânica teste estudos 10,11,12,13,14. O design específico apresentado aqui é uma adaptação de um dispositivo mecânico de teste utilizado para a realização de experiências de contato adesivo 14. Um projeto similar também tem sido usado em um micro disponível comercialmente-tribometer 15,16.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56571/56571fig1.jpg"> 
Figura 1: visão geral do dispositivo mecânico Custom-Built teste. (A), A renderização de desenho assistido por computador do dispositivo. Os componentes de fase são destacados em verde. A força de sensoriamento subconjunto (cantilever, ponto de carga (LP)) é destacada em vermelho. (B) uma ampliada vista do (A). O espelho de LP é mostrado em azul na superfície superior do LP sob o FODS e é rotulado LPM. (C) o sistema de coordenadas usado para descrever o movimento da fase de tradução. Por nivelamento thestágio e na etapa 1.9 do protocolo, o <img alt="Equation 1" src="/files/ftp_upload/56571/56571eq1.jpg"> direção feita para coincidir com o vetor normal à superfície do espelho LP. (D), um esquema da configuração de flexão de três pontos mostrando a deformação do spicule e os deslocamentos medidos <img alt="Equation 49" src="/files/ftp_upload/56571/56571eq49.jpg"> </img>, e <img alt="Equation 50" src="/files/ftp_upload/56571/56571eq50.jpg"> </img>. <a href="//ecsource.jove.com/files/ftp_upload/56571/56571fig1large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
Os recursos do dispositivo são demonstrados através da realização de testes de flexão de três pontos sobre os elementos esqueléticos da esponja marinha Euplectella Aspersório6,7. Esqueleto desta esponja é um conjunto de filamentos, chamados espículas (ver Figura 2). As espículas são ≈50 µm de espessura e são compostas principalmente de silicone 6. Espículas baseada em Biosilica são encontradas em esponjas, pertencentes às classes de Demospongiae, Homosclerophorida e Hexactinellida. Esponjas, tais como E. Aspersório, que pertencem à classe Hexactinellida também são conhecidas como "esponjas de vidro". Enquanto as espículas de esponjas de vidro são compostas principalmente de sílica, tem sido demonstrado que a sílica geralmente contém uma matriz orgânica composta ou colágeno 17,18 ou quitina 19,20 , 21. esta matriz orgânica desempenha um papel importante em sílica biomineralização 18,20. Além disso, em algumas espículas a matriz orgânica também serve como um modelo para a biomineralização de cálcio 22. Além de ser distribuída dentro da sílica, a matriz orgânica também pode formar camadas distintas que particionem sílica do spicule em lamelas concêntricas, cilíndricas 6,23. Tem sido demonstrado que essa arquitetura concêntrica, lamelar pode afetar deformação comportamento 6,7,8,24,25,26 das espículas . Por conseguinte, propriedades mecânicas das espículas são determinadas por uma combinação de sua química (i. e., a estrutura química do composto a sílica-proteína) e sua arquitetura 27. A estrutura química e a arquitetura de espículas de esponja de vidro ainda estão sob investigação de28, 24,29.
A maioria das espículas em Aspersório E. é cimentada juntos para formar uma gaiola esquelética rígida. No entanto, na base do esqueleto... há um tufo de muito tempo nas espículas (≈10 cm) conhecido como as espículas de âncora (ver Figura 2). Descrevemos o protocolo para a realização de testes de flexão de três pontos em pequenas secções de espículas a âncora.
Na etapa 1 do protocolo, é descrito o procedimento para montagem e alinhando os componentes do dispositivo de teste mecânico custom-built. As etapas 2 e 4 do protocolo fornecem instruções para gerar dados de calibração, usados para calcular as forças e deslocamentos no teste de flexão. As medidas tomadas para preparar uma seção de um spicule e montá-lo para o dispositivo de teste são descritas na etapa 3. O procedimento para a realização dos ensaios de flexão na seção spicule é descrito na etapa 5. Finalmente, na seção de Resultados de representante dos dados de calibração obtidos nos passos 2 e 4 são utilizados juntamente com os dados de teste flexão obtidos na etapa 5 para calcular <img alt="Equation 2" src="/files/ftp_upload/56571/56571eq2.jpg"> e <img alt="Equation 3" src="/files/ftp_upload/56571/56571eq3.jpg"> </img>.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56571/56571fig2.jpg"> 
Figura 2: Procedimento para corte e inspecionando espículas E. Aspersório. (A) o esqueleto de E. Aspersório. O tufo de espículas de âncora autônoma é mostrado na base do esqueleto. Barra de escala é ~ 25 mm. (B) uma spicule única âncora é mantido no lugar em uma corrediça do microscópio usando um pincel de de Marta #00000 vermelho e seccionado usando uma lâmina de barbear. Barra de escala é ~ 12 mm. (C) uma seção de um Aspersório E. spicule colocada do outro lado da trincheira, na fase de amostra. A bordas de trincheira e a trincheira ridge são destacadas em verde-azulado e laranja, respectivamente. O spicule é empurrado contra o cume de trincheira para garantir que seu eixo é perpendicular às bordas da trincheira. (D) A Micrografia de uma spicule que passa o procedimento de inspeção descrito no passo 3.4 do protocolo, que descreve como determinar se uma seção de spicule está danificada e deve ser descartada. (E) A lâmina histológica de um spicule contendo muitas rachaduras e falta de grandes seções de camadas de sílica que falhariam o procedimento de inspeção descrito no passo 3.4 do protocolo. Escala de barras = 250 µm (C), (D) de 100 µm e 100 µm (E). <a href="//ecsource.jove.com/files/ftp_upload/56571/56571fig2large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>

<table>
	<tbody>
		<tr>
			<td>TMC 36&#34; x 48&#34; isolation table with 4&#34; CleanTop breadboard</td>
			<td>TMC</td>
			<td>63-563</td>
			<td>Isolation Table</td>
		</tr>
		<tr>
			<td>Diffeential Screw Adjuster</td>
			<td>Thorlabs</td>
			<td>DAS110</td>
			<td>For stage leveling plate</td>
		</tr>
		<tr>
			<td>1&#34; Travel Micrometer Head with 0.001&#34; Graduations</td>
			<td>Thorlabs</td>
			<td>150-801ME</td>
			<td>For stage leveling plate</td>
		</tr>
		<tr>
			<td>Right-Angle Bracket for PT Series Translation Stages, 1/4&#34;-20 Mounting Holes</td>
			<td>Thorlabs</td>
			<td>PT102</td>
			<td>For microscope mount</td>
		</tr>
		<tr>
			<td>1&#34; Dovetail Translation Stage, 1/4&#34;-20 Taps</td>
			<td>Thorlabs</td>
			<td>DT25</td>
			<td>For microscope mount</td>
		</tr>
		<tr>
			<td>1&#34; Translation Stage with 1/4&#34;-170 Adjustment Screw, 1/4&#34;-20 Taps</td>
			<td>Thorlabs</td>
			<td>PT1B</td>
			<td>For microscope mount</td>
		</tr>
		<tr>
			<td>12&#34; Length, Dovetail Optical Rail</td>
			<td>Edmund Optics</td>
			<td>54-401</td>
			<td>For microscope mount</td>
		</tr>
		<tr>
			<td>2.5&#34; Width, Dovetail Carrier</td>
			<td>Edmund Optics</td>
			<td>54-404</td>
			<td>For microscope mount</td>
		</tr>
		<tr>
			<td>0.5&#34; Width, Dovetail Carrier</td>
			<td>Edmund Optics</td>
			<td>54-403</td>
			<td>For microscope mount</td>
		</tr>
		<tr>
			<td>InfiniTube Mounting C-Clamp with &#188;-20</td>
			<td>Edmund Optics</td>
			<td>57-788</td>
			<td>Microscope component</td>
		</tr>
		<tr>
			<td>Standard (with no In-Line Attachment), InfiniTube</td>
			<td>Edmund Optics</td>
			<td>56-125</td>
			<td>Microscope component</td>
		</tr>
		<tr>
			<td>Standard In-Line Attachment (Optimized at 2X-10X), InfiniTube</td>
			<td>Edmund Optics</td>
			<td>56-126</td>
			<td>Microscope component</td>
		</tr>
		<tr>
			<td>Mitutoyo/Achrovid Objective Adapter (M26 to M27)</td>
			<td>Edmund Optics</td>
			<td>53-787</td>
			<td>Microscope component</td>
		</tr>
		<tr>
			<td>5X Infinity Achrovid Microscope Objective</td>
			<td>Edmund Optics</td>
			<td>55-790</td>
			<td>Microscope component</td>
		</tr>
		<tr>
			<td>0.316&#34; ID, Fiber Optic Adapter SX-6</td>
			<td>Edmund Optics</td>
			<td>38-944</td>
			<td>Microscope component</td>
		</tr>
		<tr>
			<td>&#188;&#34; x 36&#34;, Flexible Fiber Optic Light Guide</td>
			<td>Edmund Optics</td>
			<td>42-347</td>
			<td>Microscope component</td>
		</tr>
		<tr>
			<td>115V, MI-150 Fiber Optic Illuminator w/IR Filter and Holder</td>
			<td>Edmund Optics</td>
			<td>55-718</td>
			<td>Microscope component</td>
		</tr>
		<tr>
			<td>Allied Vision Manta G-223 2/3&#34; Color CMOS Camera</td>
			<td>Edmund Optics</td>
			<td>88-452</td>
			<td>Microscope component</td>
		</tr>
		<tr>
			<td>Power Supply for Manta/ Guppy Pro/ Stingray/ Pike</td>
			<td>Edmund Optics</td>
			<td>68-586</td>
			<td>Microscope component</td>
		</tr>
		<tr>
			<td>1/4&#34; Travel Single Axis Translation Stage</td>
			<td>Thorlabs</td>
			<td>MS1S</td>
			<td>FODS micrometer</td>
		</tr>
		<tr>
			<td>Analog Reflectance Dependent Fiber Optic Displacement Sensor</td>
			<td>Philtec</td>
			<td>D20</td>
			<td>FODS</td>
		</tr>
		<tr>
			<td>30V, 3A DC Power Supply</td>
			<td>Agilent</td>
			<td>U8001A</td>
			<td>Power supply for DAQ and FODS</td>
		</tr>
		<tr>
			<td>14-Bit, 48 kS/s Low-Cost Multifunction DAQ</td>
			<td>National Instruments</td>
			<td>USB-6009</td>
			<td>DAQ for FODS</td>
		</tr>
		<tr>
			<td>Three Axis Motorized Translation Stage</td>
			<td>Thorlabs</td>
			<td>Thorlabs T25 XYZ-E/M</td>
			<td>Translation stage</td>
		</tr>
		<tr>
			<td>T-Cube DC Servo Motor Controller</td>
			<td>Thorlabs</td>
			<td>TDC001</td>
			<td>Motor controller for stage</td>
		</tr>
		<tr>
			<td>T-Cube Power Supply</td>
			<td>Thorlabs</td>
			<td>TPS001</td>
			<td>Power supply for motor controller</td>
		</tr>
		<tr>
			<td>National Instruments LabVIEW (2013 SP1)</td>
			<td>National Instruments</td>
			<td></td>
			<td>Used for running software</td>
		</tr>
		<tr>
			<td>National Instruments LabVIEW Vision Acquisition Software (2016)</td>
			<td>National Instruments</td>
			<td></td>
			<td>Used for running software</td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ci-POL Main Body</td>
			<td>MVI</td>
			<td>MDA96000</td>
			<td>Polarized light microscope</td>
		</tr>
		<tr>
			<td>Nikon Pi Intermediate Tube with Analyzer Slider</td>
			<td>MVI</td>
			<td>MDB45305</td>
			<td>Polarized light microscope</td>
		</tr>
		<tr>
			<td>Nikon Dia-Polarizer</td>
			<td>MVI</td>
			<td>MDN11920</td>
			<td>Polarized light microscope</td>
		</tr>
		<tr>
			<td>Power Cord - 7&#39;6&#34;</td>
			<td>MVI</td>
			<td>79035</td>
			<td>Polarized light microscope</td>
		</tr>
		<tr>
			<td>Nikon P-Amh Mechanical Stage</td>
			<td>MVI</td>
			<td>MDC45000</td>
			<td>Polarized light microscope</td>
		</tr>
		<tr>
			<td>Nikon Lwd Achromat Condenser</td>
			<td>MVI</td>
			<td>MBL16100</td>
			<td>Polarized light microscope</td>
		</tr>
		<tr>
			<td>Nikon LV-NBD5BD-CH Manual Quint Nosepiece ESD</td>
			<td>MVI</td>
			<td>MBP60125</td>
			<td>Polarized light microscope</td>
		</tr>
		<tr>
			<td>Nikon C-TF Trinocular Tube F</td>
			<td>MVI</td>
			<td>MBB93100</td>
			<td>Polarized light microscope</td>
		</tr>
		<tr>
			<td>Nikon CFI 10X Eyepiece FN 22mm NC</td>
			<td>MVI</td>
			<td>MAK10110</td>
			<td>Polarized light microscope</td>
		</tr>
		<tr>
			<td>Nikon TU Plan Flour BD 10x Objective</td>
			<td>MVI</td>
			<td>MUE42100</td>
			<td>Polarized light microscope</td>
		</tr>
		<tr>
			<td>Venus Flower Basket Sponge</td>
			<td>Denis Brand</td>
			<td>N/A</td>
			<td>Sponge skeleton</td>
		</tr>
		<tr>
			<td>3.5X Headband Flip-Up Magnifier</td>
			<td>McMaster Carr</td>
			<td>1490T5</td>
			<td>Used for spicule sectioning</td>
		</tr>
		<tr>
			<td>&#216;1&#34; Silicon Wafer, Type P / &#60;100&#62;</td>
			<td>Ted Pella</td>
			<td>16011</td>
			<td>Used for load point mirror</td>
		</tr>
		<tr>
			<td>Low Lint Tapered Tip Cotton Swab</td>
			<td>McMaster Carr</td>
			<td>71035T31</td>
			<td>Used for cleaning LP mirror</td>
		</tr>
		<tr>
			<td>Rubber grip precision knife</td>
			<td>McMaster Carr</td>
			<td>35575A68</td>
			<td>Used for sectioning spicules</td>
		</tr>
		<tr>
			<td>Microscope Slides, frosted end, 75 x 25 x 1mm</td>
			<td>Ted Pella</td>
			<td>260409</td>
			<td>Used for sectioning spicules</td>
		</tr>
		<tr>
			<td>Sable Brushes, #00000, 0.08mm W x 4.0mm L</td>
			<td>Ted Pella</td>
			<td>11806</td>
			<td>Used for handling spicules</td>
		</tr>
		<tr>
			<td>PELCO Pro High Precision Tweezers, extra fine tips, superior finish</td>
			<td>Ted Pella</td>
			<td>5367-5NM</td>
			<td>Used for handling spicules</td>
		</tr>
		<tr>
			<td>Dual Axis Linear Scale Micrometer</td>
			<td>Edmund Optics</td>
			<td>58-608</td>
			<td>Used for calibrating the microscopes</td>
		</tr>
		<tr>
			<td>FLEX-A-TOP FT-38 CAS</td>
			<td>ESD Plastic Containers</td>
			<td>FT-38-CAS</td>
			<td>Used for storing spicules</td>
		</tr>
		<tr>
			<td>Plastic Vial Bullseye Level</td>
			<td>McMaster Carr</td>
			<td>2147A11</td>
			<td>Used for leveling the stage</td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>Mettler Toledo</td>
			<td>MS105DU</td>
			<td>Used to mass calibration weights</td>
		</tr>
	</tbody>
</table>

a millimeter scale flexural testing system for measuring the mechanical properties of marine sponge spicules

Muitos carga rolamento estruturas biológicas (LBBSs) — como rachises pena e espículas — são pequenas (&#60; 1 mm) mas não microscópicas. Medindo o comportamento à flexão destes LBBSs é importante para compreender as origens de suas funções mecânicas notáveis.
Descreveremos um protocolo para realização de testes de flexão de três pontos usando um dispositivo de teste mecânico personalizado que pode medir forças que variam de 10-5 a 101 N e deslocamentos variando de 10-7 a 10-2 m. A principal vantagem deste dispositivo mecânico de testes é que as capacidades de força e o deslocamento podem ser facilmente ajustadas para LBBSs diferentes. Princípio de funcionamento do dispositivo é semelhante de um microscópio de força atômica. Ou seja, a força é aplicada para a LBBS por um ponto de carga que é anexado ao fim de um cantilever. O deslocamento do ponto de carga é medido por um sensor de deslocamento óptica de fibra e convertido em uma força usando a rigidez cantilever medido. Intervalo de força do dispositivo pode ser ajustado usando cantilevers de diferentes stiffnesses.
Os recursos do dispositivo são demonstrados através da realização de testes de flexão de três pontos sobre os elementos esqueléticos da esponja marinha Euplectella Aspersório. Os elementos esqueléticos — conhecido como espículas — são fibras de sílica que são aproximadamente 50 µm de diâmetro. Descrevemos os procedimentos para calibrar o aparelho de teste mecânico, as espículas de montagem em um dispositivo elétrico flexão de três pontos com uma extensão de mm ≈1.3, e realizando uma flexão de teste. Medem-se a força aplicada para o spicule e sua deflexão no local da força aplicada.

As saídas mais básicas de qualquer teste mecânico são a magnitude da força aplicada para a amostra e o deslocamento no local onde a força é aplicada. No caso de um teste de flexão de três pontos, o objetivo é obter a magnitude da força aplicada pelo LPT, <img alt="Equation 13" src="/files/ftp_upload/56571/56571eq13.jpg"> </img>e o deslocamento de seção transversal do espécime sob o LPT no <img alt="Equation 4" src="/files/ftp_upload/56571/56571eq4.jpg"> dir...

Watch this Scientific Journal Video about A Millimeter Scale Flexural Testing System for Measuring the Mechanical Properties of Marine Sponge Spicules at JoVE.com

A Millimeter Scale Flexural Testing System for Measuring the Mechanical Properties of Marine Sponge Spicules

Apresentamos um protocolo para a realização de testes de flexão de três pontos no sub milímetros fibras de escala usando um dispositivo de teste mecânico custom-built. O dispositivo pode medir forças que variam de 20 µN até 10 N e, portanto, pode acomodar uma variedade de tamanhos de fibra.

Um milímetro escala Flexural sistema de testes para medir as propriedades mecânicas de espículas de esponja marinha

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the ...

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8.4K visualizações.  Brown University. Apresentamos um protocolo para a realização de testes de flexão de três pontos no sub milímetros fibras de escala usando um dispositivo de teste mecânico custom-built. O dispositivo pode medir forças que variam de 20 µN até 10 N e, portanto, pode acomodar uma variedade de tamanhos de fibra.

Vídeo: A Millimeter Scale Flexural Testing System for Measuring the Mechanical Properties of Marine Sponge Spicules

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

Quantificar as propriedades mecânicas do Glicocálix endotelial com Microscopia de Força Atômica

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

Bioengenharia

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Projeto de um biorreator Carregando Mecânica Biaxial para Engenharia de Tecidos

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Medir as propriedades mecânicas das células vivas Usando Microscopia de Força Atômica

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

Produção Robotic da Cancer Cell Spheroids com um sistema aquoso bifásico para Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

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 ...

A Protocol for Bioinspired Design: A Ground Sampler Based on Sea Urchin Jaws

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, known as the Aristotle's lantern, is a compelling source of bioinspiration with an intricate network of musculature and calcareous teeth that can scrape, cut, chew food and bore holes into rocky substrates. We describe the bioinspiration process as including animal observation, specimen characterization, device fabrication and mechanism bioexploration. The last step of bioexploration allows for a deeper understanding of the initial biology. The design architecture of the Aristotle's lantern is analyzed with micro-computed tomography and individual teeth are examined with scanning electron microscopy to identify the microstructure. Bioinspired designs are fabricated with a 3D printer, assembled and tested to determine the most efficient lantern opening and closing mechanism. Teeth from the bioinspired lantern design are bioexplored via finite element analysis to explain from a mechanical perspective why keeled tooth structures evolved in the modern sea urchins we observed. This circular approach allows for new conclusions to be drawn from biology and nature.

A protocol for bioinspired design is described for a sampling device based on the jaws of a sea urchin. The bioinspiration process includes observing the sea urchins, characterizing the mouthpiece, 3D printing of the teeth and their assembly, and bioexploring the tooth structure.

Um protocolo para Bioinspirada Projeto: Um Sampler terra Baseado em Sea Urchin Jaws

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, ...

Three-Dimensionally Printed Microfluidic Cross-flow System for Ultrafiltration/Nanofiltration Membrane Performance Testing

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane technology. Understanding the fouling process could lead to optimization and higher efficiency of membrane based filtration. Here we show the design and fabrication of an automated three-dimensionally (3-D) printed microfluidic cross-flow filtration system that can test up to 4 membranes in parallel. The microfluidic cells were printed using multi-material photopolymer 3-D printing technology, which used a transparent hard polymer for the microfluidic cell body and incorporated a thin rubber-like polymer layer, which prevents leakages during operation. The performance of ultrafiltration (UF), and nanofiltration (NF) membranes were tested and membrane fouling could be observed with a model foulant bovine serum albumin (BSA). Feed solutions containing BSA showed flux decline of the membrane. This protocol may be extended to measure fouling or biofouling with many other organic, inorganic or microbial containing solutions. The microfluidic design is especially advantageous for testing materials that are costly or only available in small quantities, for example polysaccharides, proteins, or lipids due to the small surface area of the membrane being tested. This modular system may also be easily expanded for high throughput testing of membranes.&#160;

Design and fabrication of a three-dimensionally (3-D) printed microfluidic cross-flow filtration system is demonstrated. The system is used to test performance and observe fouling of ultrafiltration and nanofiltration (thin film composite) membranes.

Tridimensionalmente Impresso Microfluidic Sistema Cross-flow para ultrafiltração / nanofiltração membrana Teste de Desempenho

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane ...

A Performance-testing Platform for a Conduction Micropump with an FR-4 Copper-clad Electrode Plate

Here, a conduction micropump with symmetric planar electrode pairs prepared on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) is fabricated. It is used to investigate the influence of chamber dimensions on the performance of a conduction micropump and to determine the reliability of the conduction pump when acetone is used as the working fluid. A testing platform is set up to evaluate conduction micropump performance under different conditions. When the chamber height is 0.2 mm, the pump pressure reaches its peak value.

This paper presents a protocol for the fabrication of a conduction micropump using symmetric planar electrodes on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) to test the influence of chamber dimensions on the performance of a conduction micropump.

Uma plataforma de testes de desempenho para uma microbomba de condução com uma placa de eletrodos de cobre-folheada FR-4

Here, a conduction micropump with symmetric planar electrode pairs prepared on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) is ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

Rígida incorporação de fixo e manchado, inteiro, espécimes de milímetro-escala para histologia seção livre 3D por Microtomografia computadorizada

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Automated Counterflow Centrifugal System for Small-Scale Cell Processing

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and product concentration are essential components for most manufacturing processes. However, at the early stages of product development, these steps are often performed manually. Manual dead-end centrifugation for buffer exchange is labor-intensive, costly, and not scalable. A closed automated system can effectively eliminate this laborious step, but implementation can be challenging. Here, we describe a newly developed cell processing device that is suitable for small- to medium-scale cell processing and aims to bridge the gap between manual processing and large-scale automation. This protocol can be easily applied to various cell types and processes by modifying the flow rate and centrifugation speed. Our protocol demonstrated high cell recovery with shorter processing times in comparison to the manual process. Cells recovered from the automated process also maintained their proliferation rates. The device can be applied as a modular component in a closed manufacturing process to accommodate steps such as buffer exchange, cell formulation, and cryopreservation.

Automation is key to upscaling and cost management in cell manufacturing. This manuscript describes the use of a counterflow centrifugal cell processing device for automating the buffer exchange and cell concentration steps for small-scale bioprocessing.

Sistema centrífuga de contrafluxo automatizado para processamento de células em pequena escala

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and ...