Y. T. Y. gostaria de reconhecer o apoio do Ministério da ciência e tecnologia sob concessão números mais 105-2221-E-007-MY3 e da National Tsing Hua University sob concessão números 105N518CE1 e 106N518CE1 financeiro.

1. Setup óptico Nota: O princípio da instalação do óptico é ilustrado na Figura 1.
Pinça <ol><li>conjunto até o óptico kit (veja a Tabela de materiais) e o módulo de fluorescência (ver Tabela de materiais) como por seus manuais. Conectar uma fonte de 470 nm azul luz emitindo luz de diodo (LED) para o módulo fluorescente.</li>
	<li>Substituir a alta abertura numérica (NA) (at = 1.25, ampliação 100x) objectivo de imersão de óleo por um trabalho de longo distância objetivo do microscópio (WD) (comprimento focal 3,6 mm, WD = 10,6 mm, at = 0,5).</li>
	<li>Remover a lente na seção expansão de feixe do kit montado para alcançar solto com foco de raio laser.</li>
	<li>Vez na fonte de alimentação e a corrente para o laser de diodo de nm de comprimento de onda 980 e uso o carregado acoplado a câmera do dispositivo (CCD) para certificar-se de que o feixe de laser é alinhado corretamente. 
	Nota: Se o feixe de laser é bem alinhado, a câmera CCD lerá um ponto Gaussian.</li>
</ol> 2. Nanofabricação fabricação de marcador de <ol><li>. 
	Nota: Marcadores irão ajudar a posicionar a matriz de nanoplasmonic durante o processo de fabricação e o experimento de armadilhagem subsequentes. O processo detalhado é ilustrado na Figura complementar 1.
	<ol><li>Filme de óxido de estanho (ITO) de índio depósito 40 nm em uma lamela de espessura 0,17 mm com pulverização catódica. 
		Nota: O filme ITO ajudará a descarga de elétrons durante o processo de litografia e-feixe subsequentes.</li>
		<li>Spin revestir uma camada de 8 µm de fotorresiste positivo com rotação velocidade 4000 rpm e tempo de 30 s, com um aplicador de rotação.</li>
		<li>Soft cozer a amostra a 90 ° C por 5 min e alinhar a amostra com a Fotomáscara para marcador e expor a amostra à luz para 80 UV s no alinhador de máscara.</li>
		<li>Embeber a amostra no desenvolvedor fotorresiste por 130 s.</li>
		<li>Depositar uma camada de 2 nm de crómio e uma camada de 40 nm de ouro para a amostra usando evaporação térmica. 18</li> <li>mergulhar a amostra em acetona e colocá-lo em um líquido de limpeza ultra-sônico que operam em 43 kHz e 150 W por 5 min para fora do elevador</li>
	</ol></li> <li>Fabricação de matriz Nanoplasmonic <ol><li>casaco Spin uma camada de e-feixe resistir PMMA 120K com centrifugação 5000 rpm por 30 s em um aplicador de rotação. Asse a amostra a 160 ° C por 3 min em uma chapa quente.</li>
		<li>Spin revestir outra camada de resistir e-feixe PMMA 960K com centrifugação 5000 rpm por 30 s em um aplicador de rotação. Asse a amostra a 160 ° C por 3 min em uma chapa quente.</li>
		<li>Escritor e-feixe de uso para expor o feixe e resistir com aceleração tensão 30 kV e dosagem 400 C/cm 2.</li>
		<li>Depositar uma camada de 40 nm de ouro em um evaporador térmico.</li>
		<li>Mergulhar a amostra em acetona e colocá-lo em um líquido de limpeza ultra-sônico por 5 min para fora do elevador</li>
	</ol></li></ol> 3. Exemplo de sistema de resfriamento e a calibração de temperatura Nota: A amostra refrigerar cenografia é mostrada na complementar Figura 2.
<ol><li>Fazendo o circuito controlador para refrigeração de amostra <ol><li>Coloque os resistores, transistores de junção bipolar e transistores de efeito de campo de óxido de metal de poder na placa de circuito personalizada seguindo o diagrama de circuito da complementar Figura 3 . Todos esses componentes com ferro de solda de solda.</li>
		<li>Fios de conexão entre a porta de controle da placa de circuito e a placa de controle eletrônico. Ligue os fios entre a porta de saída da placa de circuito e resfriamento termoelétrico (TEC) elemento. Coloque o elemento TEC no palco a amostra com o calor a afundar-se. 
		&#8203; Nota: TEC o elemento tem um buraco no centro para permitir que o feixe de laser passar.</li>
		<li>Fios de conexão da placa de circuito para 5 V de alimentação. Use a câmera infravermelha prospectiva para monitorar a temperatura para verificar se o resfriamento termoelétrico é corretamente refrigerar para baixo.</li>
	</ol></li> <li>Calibração da temperatura medida na prospectivas câmera e resistência temperatura detector (IDT) termômetro infravermelho.
	<ol><li>Coloque o Termómetro RTD em uma lamela em branco e aplique uma pequena quantidade de pasta térmica para assegurar o adequado contato térmico entre o Termómetro RTD e lamela.</li>
		<li>Alterar o ajuste da potência de saída do circuito de controle eletrônico para elemento TEC, alterando o ciclo de trabalho de configuração de modulação de largura de pulso e esperar por 3 min certificar-se de que atingir a temperatura de estado estacionário. Leia a temperatura com o termômetro de IDT.</li>
		<li>Vez na câmera infravermelha olhando para a frente e monitor da temperatura. Repita isto em várias configurações de potência de saída para obter a curva de calibração de temperatura. Uma curva de calibração de temperatura representativa é mostrada na Figura complementar 4. 
		Nota: É fundamental fazer calibração entre RTD termômetro e a câmera de infravermelha olhando para a frente porque a leitura da temperatura da câmara de infravermelha voltada para o futuro deve ser exactos para garantir a temperatura correta é alcangada.</li>
	</ol></li></ol> 4. Interceptação de micropartículas de <ol><li>dilua micro partículas de poliestireno do diâmetro 2 µm em água deionizada em um microcentrifugetube de relação volume apropriado. 
	Nota: A concentração de micro partículas pode ser ajustada de acordo com o objetivo do experimento. Enquanto menor concentração permite um intervalo de tempo de amostra entre eventos de armadilhagem única partícula, maior concentração encurtará o tempo para a captura de partículas múltiplas. Para a captura de partícula única, uma concentração típica é ~0.05% (w/v).</li>
	<li>Colocar a amostra com a matriz nanoplasmonic no palco e ligar um 470 nm LED como fonte de luz de fluorescência e definir manualmente a potência de 5 mW para a imagem latente de campo brilhante.</li>
	<li>Use o marcador para localizar a matriz nanoplasmonic, alinhe a amostra e usar a câmera do CCD para certificar-se de que a matriz está no centro da região de interesse na tela do computador.</li>
	<li>Dispense 10 µ l das diluídos micro partículas de diâmetro 2 µm na amostra com uma pipeta micro.</li>
	<li>Ligar a alimentação em corrente para o diodo de nm de comprimento de onda 980 para excitar a ressonância plasmônico da matriz com um poder no intervalo ~ 1 mW a 10 mW.</li>
	<li>Manualmente, ligue a fonte de alimentação para a placa de controle eletrônico para resfriar a amostra para um estado constante de temperatura ~ 4 ° C.</li>
	<li>No software visualizador, clique o &#34; gravar vídeo &#34; sequência para abrir a caixa de diálogo de gravação. Clique o &#34; registro &#34; botão para iniciar o vídeo 1.5 do movimento das partículas de micro, com uma taxa de quadros de 10 quadros/s de gravação da amostra sob a influência do feixe de laser, usando a câmera do CCD. Clique o &#34; parar de &#34; o botão para parar a gravação. Veja o vídeo 1.</li>
</ol>

<ol>
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Os autores não têm nada para divulgar.

O procedimento descrito aqui permite que o leitor reproduzir confiantemente armadilhas em uma base diária. Uma orientação empírica geral para projetar um lattice ótico utilizável é usar um tamanho comparável para plasmônico nanoarray, distância interdisc e preso o tamanho de partícula. Comparado a um nanostructure plasmônico único, isolado, o projeto de estrutura óptica em conjunto com a alta potência óptica proporcionada pelo resfriamento da amostra a ~ 4 ° C, usado aqui grandemente aumenta a probabilid...

A captura óptica de partículas micro escala foi originalmente desenvolvida por Arthur Askin na década de 1970. Desde a sua invenção, a técnica foi desenvolvida como uma ferramenta versátil para micro e nanomanipulação1,2. Convencional óptico prendendo com base no campo distante focar princípio inerentemente é limitada pela difração em seu confinamento espacial, onde a força de armadilhagem diminui drasticamente (seguir um ~umalei de3 de uma partícula de raio um) 3. para superar tais limites de difração, pesquisadores desenvolveram técnicas de armadilhagem óptica de campo próximo, com base no campo evanescente óptico usando nanoestruturas metálicas plasmônico e, além disso, a captura de nanoescala objetos para baixo para moléculas de proteína única tem sido demonstrada4,5,6,7,8,9,10,11. Além disso, o retículo óptico plasmônico é criado a partir de matrizes de nanoestruturas plasmônico periódicas para conferir o transporte de longo alcance do micro e nanopartículas e múltiplas partículas empilhamento11,12. Um grande obstáculo para interromper a captura em um lattice ótico é convecção fototérmicos e foram envidados esforços para elucidar seus efeitos por vários grupos14,15,16,17. Usando a função de Green, Baffou et al ter calculado um perfil de temperatura por cada nanostructure plasmônico como um aquecedor de ponto de modelagem e então experimentalmente validados seu modelo14. Grupo do Toussant também mediu a convecção induzida por plasmon com partícula velocimetry15. Grupo do autor também tem caracterizado o transporte campo próximo e convecçao e demonstrou uma estratégia de engenharia para suprimir originando convecção16,17.
Aqui apresentamos o projeto de uma instalação de ótica e um procedimento detalhado especificamente para experimentos de armadilhagem plasmônico retículo óptico. O potencial óptico foi criado por iluminando uma matriz de ouro nanodiscs com um feixe gaussiano vagamente concentrado. Um esquema para suprimir a convecção originando pelo resfriamento da amostra a uma temperatura baixa (~ 4 ° C) para a captura ideal é também descrever aqui17. Sob a aproximação de Boussinesq, uma ordem de magnitude estimada da convecção natural velocidade u é dada por u ~L2 gβΔT / v, onde L é a escala de comprimento da fonte de calor e Δ T é o aumento de temperatura em relação a referência devido ao aquecimento.  g e β são a aceleração gravitacional e coeficiente de expansão térmica, respectivamente. Em temperaturas perto de 4 ° C, a densidade do meio de água apresenta dependência de temperatura anômalas e isso se traduz em um coeficiente de expansão térmica próximo de zero e, portanto, uma convecção originando muito pequeno.

<table style="border-collapse:collapse;width:629pt" width="839">
	<colgroup>
		<col style="width:207pt" width="276" />
		<col style="width:131pt" width="174" />
		<col style="width:122pt" width="162" />
		<col style="width:170pt" width="227" />
	</colgroup>
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Thermoelectric cooling element</td>
			<td class="xl69" style="width:131pt" width="174">Thorlabs</td>
			<td class="xl69" style="width:122pt" width="162">TEC 1.4-6</td>
			<td class="xl70" style="width:170pt" width="227">TEC element for sample cooling</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl69" height="42" style="width:207pt;height:31.5pt" width="276">RTD thermometer</td>
			<td class="xl69" style="width:131pt" width="174">Omega Engineering</td>
			<td class="xl69" style="width:122pt" width="162">RTD Thermometer 969C</td>
			<td class="xl70"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Forward looking infrared camera</td>
			<td class="xl69" style="width:131pt" width="174">FLIR&#160;</td>
			<td class="xl69" style="width:122pt" width="162">FLIR One</td>
			<td class="xl70">IR camera for temperature monitoring</td>
		</tr>
		<tr height="46" style="height:34.5pt">
			<td class="xl69" height="46" style="width:207pt;height:34.5pt" width="276">light emitting diode light source</td>
			<td class="xl69" style="width:131pt" width="174">Touchbright</td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl69" style="width:170pt" width="227">Light source for illumination for fluorescent imaging</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Long working distance objective</td>
			<td class="xl69" style="width:131pt" width="174">Olympus</td>
			<td class="xl69" style="width:122pt" width="162">LMPLFLN</td>
			<td class="xl70">For illuminating the sample and imaging</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Optical trap kit</td>
			<td class="xl69" style="width:131pt" width="174">Thorlabs</td>
			<td class="xl69" style="width:122pt" width="162">OTKB/M</td>
			<td class="xl70"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl71" height="21" style="height:15.75pt">Cover slip</td>
			<td class="xl69" style="width:131pt" width="174"></td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl69" style="width:170pt" width="227">thickness 0.17 mm</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl71" height="21" style="height:15.75pt">Scanning electron microscope</td>
			<td class="xl69" style="width:131pt" width="174">Hitachi</td>
			<td class="xl69" style="width:122pt" width="162">SEM-Hitachi S3400N</td>
			<td class="xl69" style="width:170pt" width="227"></td>
		</tr>
		<tr height="44" style="height:33.0pt">
			<td class="xl71" height="44" style="height:33.0pt">Electron beam blanker</td>
			<td class="xl69" style="width:131pt" width="174">DEBEN</td>
			<td class="xl69" style="width:122pt" width="162">PCD beam blanker</td>
			<td class="xl69" style="width:170pt" width="227">the blanker is added to the scanning electron microscope&#160;</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl71" height="21" style="height:15.75pt">Thermal evaporator</td>
			<td class="xl69" style="width:131pt" width="174">SYSKEY Technology</td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl69" style="width:170pt" width="227"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl71" height="21" style="height:15.75pt">Mask aligner</td>
			<td class="xl69" style="width:131pt" width="174">Karl Suss</td>
			<td class="xl69" style="width:122pt" width="162">MJB 3</td>
			<td class="xl69" style="width:170pt" width="227">For marker fabrication</td>
		</tr>
		<tr height="24" style="height:18.0pt">
			<td class="xl69" height="24" style="width:207pt;height:18.0pt" width="276">Electron beam resist</td>
			<td class="xl69" style="width:131pt" width="174">Sigma Alrich</td>
			<td class="xl69" style="width:122pt" width="162">PMMA 120K</td>
			<td class="xl69" style="width:170pt" width="227">For e-beam lithography</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Electron beam resist</td>
			<td class="xl69" style="width:131pt" width="174">Sigma Alrich</td>
			<td class="xl69" style="width:122pt" width="162">PMMA 960K</td>
			<td class="xl70">For e-beam lithography</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl69" height="42" style="width:207pt;height:31.5pt" width="276">Fluoresent labeled polystyrene microspheres</td>
			<td class="xl69" style="width:131pt" width="174">Polyscience</td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl70">2 um diameter</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Bipolar transistor</td>
			<td class="xl69" style="width:131pt" width="174">Mouser</td>
			<td class="xl69" style="width:122pt" width="162">2N3904</td>
			<td class="xl70">quantity 2 for TEC driver circuit</td>
		</tr>
		<tr height="28" style="height:21.0pt">
			<td class="xl69" height="28" style="width:207pt;height:21.0pt" width="276">Bipolar transistor</td>
			<td class="xl69" style="width:131pt" width="174">Mouser</td>
			<td class="xl69" style="width:122pt" width="162">2N3906</td>
			<td class="xl70">quantity 2 for TEC driver circuit</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">MOSFET power transistor</td>
			<td class="xl69" style="width:131pt" width="174">Mouser</td>
			<td class="xl69" style="width:122pt" width="162">IRF5305</td>
			<td class="xl70">quantity 2 for TEC driver circuit</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">MOSFET power transistor</td>
			<td class="xl69" style="width:131pt" width="174">Mouser</td>
			<td class="xl69" style="width:122pt" width="162">IRF131ON</td>
			<td class="xl70">quantity 2 for TEC driver circuit</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">10 kOhm resistor</td>
			<td class="xl69" style="width:131pt" width="174">Mouser</td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl70">quantity 6 for TEC driver circuit</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">910 Ohm resistor</td>
			<td class="xl69" style="width:131pt" width="174">Mouser</td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl70">quantity 2 for TEC driver circuit</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Photoresist</td>
			<td class="xl69" style="width:131pt" width="174">Microchemicals</td>
			<td class="xl69" style="width:122pt" width="162">AZ4620</td>
			<td class="xl70">For marker fabrication</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Acetone</td>
			<td class="xl69" style="width:131pt" width="174">Sigma Alrich</td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl70">For marker fabrication</td>
		</tr>
		<tr height="41" style="height:30.75pt">
			<td class="xl69" height="41" style="width:207pt;height:30.75pt" width="276">Fluorescence Module for the OTKB/M, Metric Threads</td>
			<td class="xl69" style="width:131pt" width="174">Thorlabs</td>
			<td class="xl69" style="width:122pt" width="162">OTKB-FL/M</td>
			<td class="xl69" style="width:170pt" width="227"></td>
		</tr>
		<tr height="49" style="height:36.75pt">
			<td class="xl69" height="49" style="width:207pt;height:36.75pt" width="276">Fluorescent filter set</td>
			<td class="xl69" style="width:131pt" width="174">Thorlabs</td>
			<td class="xl69" style="width:122pt" width="162">MDF-FITC</td>
			<td class="xl70">For Fluorescein Isothiocyanate (FITC)</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl70" height="21" style="height:15.75pt">Ultrasonic cleaner</td>
			<td class="xl69" style="width:131pt" width="174">Delta</td>
			<td class="xl70">DC150H</td>
			<td class="xl70">For the lift off step</td>
		</tr>
	</tbody>
</table>

trapping of micro particles in nanoplasmonic optical lattice

A Pinça óptica plasmônico foi desenvolvida para superar os limites de difração da pinça óptica convencional de campo distante. Plasmônico óptica treliça consiste de uma matriz de nanoestruturas, que exibem uma variedade de armadilhas e comportamentos de transporte. Nós relatamos os procedimentos experimentais para capturar micropartículas em um lattice ótico nanoplasmonic quadrado simples. Também descrevemos a configuração óptica e a nanofabricação de uma matriz de nanoplasmonic. O potencial óptico é criado por iluminando uma matriz de nanodiscs de ouro com um feixe gaussiano de 980 nm comprimento de onda e ressonância de plasmon emocionante. O movimento das partículas é monitorado por imagens de fluorescência. Um esquema para suprimir originando convecção também é descrito para aumentar a potência óptica utilizável para interceptação de ideal. Supressão da convecção é alcançado pelo resfriamento da amostra a uma temperatura baixa e utilizando o coeficiente de expansão térmica próximo de zero de um meio de água. Tanto transporte de partícula única e múltiplas armadilhas de partícula são relatados aqui.

Trajetórias de partícula única foram gravadas por uma câmera CCD na nossa experiência e as imagens foram processadas com um programa personalizado para extrair trajetória16 do cada partícula. Resultados representativos são exibidos na Figura 3 e 1 vídeo para microesferas com diâmetros de 2 µm. foram observadas várias armadilhas de partícula dentro lattice ótico. Imagens sucessivas, extraídas de um vídeo...

Watch this Scientific Journal Video about Trapping of Micro Particles in Nanoplasmonic Optical Lattice at JoVE.com

Trapping of Micro Particles in Nanoplasmonic Optical Lattice

Descrevemos um procedimento para interceptar opticamente micropartículas em nanoplasmonic lattice ótico.

Captura de Micro partículas em Lattice ótico Nanoplasmonic

The plasmonic optical tweezer has been developed to overcome the diffraction limits of the conventional far field optical tweezer. Plasmonic optical ...

trapping-of-micro-particles-in-nanoplasmonic-optical-lattice

Research

JoVE Journal

Bioengineering

6.5K visualizações.  National Tsing Hua University. Descrevemos um procedimento para interceptar opticamente micropartículas em nanoplasmonic lattice ótico.

Vídeo: Trapping of Micro Particles in Nanoplasmonic Optical Lattice

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Tridimensional Optical resolução Microscopia Fotoacústica

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Bioengenharia

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Multiparamétrico Mapping Optical do Coração Coelho Langendorff-perfundidos

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

Mapeamento de potenciais de ação óptica e transientes de cálcio no coração de rato

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

A Cre-Lox Abordagem Recombinação P para Detecção de fusão celular In Vivo</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

Em síntese in vitro de Nativo, Espaçamento fibroso Long e Colágeno Espaçamento segmentar Longo

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to give an accurate velocity profile. A protocol is presented for &mu;PIV measurements of blood flows in microchannels. At the scale of the microcirculation, blood cannot be considered a homogeneous fluid, as it is a suspension of flexible particles suspended in plasma, a Newtonian fluid. Shear rate, maximum velocity, velocity profile shape, and flow rate can be derived from these measurements. Several key parameters such as focal depth, particle concentration, and system compliance, are presented in order to ensure accurate, useful data along with examples and representative results for various hematocrits and flow conditions.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.

Micro-partículas Velocimetry Imagem para a velocidade Medidas de fluxos de sangue Micro Perfil

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to ...

Intra-lymph Node Injection of Biodegradable Polymer Particles

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these foreign molecules to T and B lymphocytes. Lymph nodes have thus become critical targets for new vaccines and immunotherapies. A recent strategy for targeting these tissues is direct lymph node injection of soluble vaccine components, and clinical trials involving this technique have been promising. Several biomaterial strategies have also been investigated to improve lymph node targeting, for example, tuning particle size for optimal drainage of biomaterial vaccine particles. In this paper we present a new method that combines direct lymph node injection with biodegradable polymer particles that can be laden with antigen, adjuvant, or other vaccine components. In this method polymeric microparticles or nanoparticles are synthesized by a modified double emulsion protocol incorporating lipid stabilizers. Particle properties (e.g.&#160;size, cargo loading) are confirmed by laser diffraction and fluorescent microscopy, respectively. Mouse lymph nodes are then identified by peripheral injection of a nontoxic tracer dye that allows visualization of the target injection site and subsequent deposition of polymer particles in lymph nodes. This technique allows direct control over the doses and combinations of biomaterials and vaccine components delivered to lymph nodes and could be harnessed in the development of new biomaterial-based vaccines.

Lymph nodes are the immunological tissues that orchestrate immune response and are a critical target for vaccines. Biomaterials have been employed to better target lymph nodes and to control delivery of antigens or adjuvants. This paper describes a technique combining these ideas to inject biocompatible polymer particles into lymph nodes.

Injeção de linfonodo intra-linfático de partículas de polímeros biodegradáveis

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these ...

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; ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

Targeted Plasma Membrane entrega de um hidrofóbica Carga encapsulado em um cristal líquido nanopartículas Transportador

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.