Os autores agradecem leitura atenta Dr. Lynnea Brumbaugh do manuscrito. Este trabalho foi patrocinado pelo National Institutes of Health Grants EB000712 R01, R01 EB008085, R01 CA134539, U54 CA136398 e 5P60 DK02057933. Prof Lihong V. Wang tem um interesse financeiro em Inc., Microphotoacoustics, Inc. e Endra, que, no entanto, não apoiar este trabalho.

1. Configuração do sistema <ol><li> Irradiação óptica <ol><li> Fonte de radiação óptica: um diodo-bombeada de estado sólido pulsado laser (INNOSLAB, Edgewave) e um laser de corante (CBR-D, Sirah). </li><li> A saída do feixe laser (largura de pulso: 7 ns) é focalizado por uma lente condensadora (LA1131, Thorlabs) para passar através de uma pinhole 50 mícrons (P50C, Thorlabs). </li><li> O pinhole é posicionado um pouco longe do foco da lente condensadora para coincidir com o diâmetro pinhole com o diâmetro do feixe fundamentais de modo eficaz para a filtragem espacial. </li><li> O feixe filtrado é atenuado por um filtro de densidade neutra (NDC-50C-2M, Thorlabs) e, em seguida, acoplada em uma fibra óptica monomodo (P1-460A-FC-2, Thorlabs). </li><li> A saída de fibra enche a abertura traseira de uma objetiva de microscópio (RMS4X, Thorlabs) para atingir um foco de difração limitada óptica de ~ 2,6 mM no comprimento de onda de 570 nm. </li></ol></li><li> Detecção ultra-sônica <ol><li> Transdutor de ultra-som: 50-MHz centrais freqüência (V214-BB-RM, Olympus-NDT). </li><li> O transdutor de ultra-som é acoplado a um home-made combinador de feixe acústico-ótico 11 para detecção ultra-sônica, que está alinhada coaxialmente com a irradiação de difração limitada óptica. </li><li> Uma cavidade esférica é um terreno fora do fundo do combinador para produzir uma lente acústica. Esta lente acústica tem uma abertura numérica de 0,5 em água e dá um diâmetro acústico focal de 43 mM na freqüência central de 50 MHz. </li><li> Os focos visuais e acústicos estão alinhados confocally para maximizar a sensibilidade de detecção. </li></ol></li><li> Acoplamento acústico <ol><li> Acoplamento ultra-sônico seca é empregada para evitar a submersão em água animais experimentais, que foi utilizado no início de sistemas de imagem fotoacústica 12. </li><li> Uma janela de imagem é aberta no fundo de uma placa de Petri (9 cm de diâmetro) e é selado com uma membrana de polietileno de ultra-som e opticamente transparentes. </li><li> Gel ultra-sônico (Image Clear, SonoTech) entre a membrana de polietileno e do objeto a ser fotografado casais a onda gerada fotoacústica do objeto para a placa de Petri, e água deionizada na placa de Petri casais ainda mais a onda até a cabeça submersa imagem OR-PAM . </li></ol></li><li> Eletrônica <ol><li> O sinal fotoacústico detectada pelo transdutor de ultra-som é amplificado por dois amplificadores em cascata (ZFL 500LN, Mini-Circuits) </li><li> O sinal amplificado é digitalizada por uma aquisição de dados 14-bit (DAQ) bordo (CompuScope 14200, Gage Ciências Aplicadas) a uma taxa de amostragem de 200 MS / s. </li></ol></li><li> Digitalização esquema <ol><li> Bidimensional (2-D) de varredura da cabeça de digitalização de imagens OR-PAM ao longo do plano (xy) horizontal é controlada por um computador pessoal, o que desencadeia tanto na placa DAQ eo laser da bomba. O sinal de disparo é sincronizado com o sinal de clock-out da placa DAQ. </li><li> O eixo rápido do scanner 2-D é definida como a direção da varredura transversal (B-scan). </li><li> Uma seqüência de B-scan imagens podem ser adquiridas por traduzir a imagem de cabeça ao longo do eixo lento para formar uma imagem volumétrica, que pode ser visto tanto em uma renderização 3-D direta ou em uma projeção 2-D amplitude máxima (MAP) de imagem . </li></ol></li></ol> 2. Alinhamento do sistema <ol><li> Use pulso-eco ultra-som e um refletor ultra-som para determinar a posição do plano focal acústica (ou seja, o tempo de atraso do sinal de gatilho para o sinal de pulso-eco ultra-som no máximo). Este passo é necessário para ser realizada apenas uma vez quando a construção do sistema de OR-PAM. </li><li> Maximizar a eficiência de acoplamento da fibra monomodo. </li><li> Aplique gel ultra-som em cima de um objeto opticamente absorvente (por exemplo, um pedaço de fita preta) e anexá-lo delicadamente debaixo da janela de imagem em placa de Petri com água deionizada. </li><li> Baixar a cabeça de imagens na água, e remover as bolhas de ar presas sob a lente acústica. </li><li> Ajustar a cabeça de imagem até que o sinal fotoacústico do objeto absorver é a partir do plano focal acústico, que pode ser julgado a partir do atraso acústico. </li><li> Ajustar a posição vertical (ou seja, a posição z) da objetiva do microscópio para maximizar a amplitude do sinal fotoacústico gerado a partir do objeto plano. A amplitude do sinal maximizada sugere que o foco óptico está alinhado com o foco acústica na direção vertical. </li><li> Ajustar as posições horizontal (posições ou seja, x, y) da objetiva do microscópio até que o sinal fotoacústico gerado a partir do destino mostra um padrão simétrico. A simetria sugere que o foco óptico está alinhado com o foco acústica na direção horizontal. </li><li> Repita os passos 2.6 e 2.7 até que o sinal fotoacústico é otimizado em ambos os simetria e amplitude. </li></ol> 3. A sa<html> procedimento experimental mple-in vivo OR-PAM do mouse ouvido vasculatura <ol><li> Esta etapa não é necessária para ratos nude. Anestesiar o animal com uma injeção intraperitoneal de um cocktail [receita: 1 ml de ketamina (100 mg / ml), 0,1 ml de xilazina (100 mg / ml), e 8,9 ml de soro; dosagem: 0,1 ml/10 g]. Raspar o cabelo no ouvido, e ainda depilar os cabelos residual com Surgi Cream (Categoria #: 82565, American International Industries) antes de limpá-lo com água deionizada. Note-se que tais depilação pode irritar um pouco a vascularização da pele e, assim, é melhor realizada 24 horas antes do experimento planejado. </li><li> Ligue o sistema a laser fotoacústica, e verificar o alinhamento do sistema. </li><li> Anestesiar o rato com 3% de isoflurano vaporizado pelo gás inalação (a taxa de fluxo típico é 1,0-1,5 l / min, dependendo do peso corporal do animal), e manter a anestesia com isoflurano a 1% durante todo o experimento. Médico-grade do ar é recomendada como o gás de inalação para manter o mouse no estado fisiológico normal. </li><li> Transferir o mouse para um estágio estereotáxica, e controlar a sua temperatura corporal a 37 ° C com uma almofada de aquecimento. </li><li> Achatar o ouvido do rato sobre uma placa de plástico e aplique uma camada de gel de ultra-som em cima da orelha. Evite bolhas de interceptação no interior do gel. Em seguida, coloque o ouvido na janela de imagem e lentamente eleve a fase animal até os contatos de ultra-som gel na parte inferior da membrana de polietileno. De contato gelatinosas é necessário porque pressionar a orelha contra a membrana pode afetar o fluxo de sangue no ouvido. </li><li> Pinça um oxímetro de pulso para a perna do mouse ou a cauda para monitorar seu estado fisiológico e aplique pomada para os olhos para evitar ressecamento e danos acidentais a laser para os olhos mouse. </li><li> Baixar a cabeça de imagens até a lente acústica é imerso em água deionizada, e remover as bolhas de ar presas sob a lente acústica. </li><li> Verificar a influência do laser para se certificar de que está dentro dos padrões de laser de segurança da American National Standards Institute 13. A influência do laser não deve exceder 20 mJ / cm 2, que se traduz em 80 nJ de laser de pulso de energia quando o feixe de laser é focado em 150 M abaixo da superfície da pele. </li><li> Definir a laser para o modo de external-gatilho e começar a digitalizar julgamento. Ajustar a posição z da cabeça de imagem até o mais forte sinal fotoacústico é a partir do plano focal acústico. </li><li> Conjunto correto parâmetros de digitalização e começar a aquisição de imagem formal. </li><li> Após a experiência, desligue o laser, levante a cabeça de imagem para fora da água deionizada, menor o estágio animal para soltar o mouse, limpar o ouvido do rato com água deionizada, desligue o sistema de anestesia eo controlador de temperatura, e descarregar o mouse desde a fase de estereotáxica. </li><li> Se imagens repetitivas é necessário, coloque o mouse em uma incubadora com a temperatura ambiente a 37 ° C. Devolver o mouse para a instalação de animais depois que acorda naturalmente. Caso contrário, siga os protocolos animal Eutanásia e descartá-la. </li></ol> 4. Funcional ou-PAM de concentração total e saturação de oxigênio da hemoglobina <ol><li> Oxihemoglobina (HBO 2) e desoxihemoglobina (HBR) são as duas principais formas de hemoglobina, a fonte endógena fotoacústica predominante na faixa visível do espectro. HbO 2 e HBR têm espectros de absorção óptica distinta e, portanto, podem ser espectralmente diferenciadas para quantificar tanto a concentração total (HBT) e saturação de oxigênio (SO 2) de hemoglobina 5. Aqui estão duas diretrizes para ajudar a selecionar comprimentos de onda ópticos adequada para o SO 2 medidas: </li><li> Comprimentos de onda deve ser selecionado dentro do Q banda do espectro de absorção da hemoglobina (ou seja, 550-600 nm) para garantir uma relação sinal-ruído suficiente e uma penetração adequada. </li><li> Comprimentos de onda onde os coeficientes de absorção da HBO 2 e HBR tem uma diferença acentuada (por exemplo, HBR-561 nm dominante e HBO 2-dominante 578 nm) são recomendados. </li></ol> Além de SO 2, HBT pode ser calculado pela adição [HBR] e [HbO 2] juntos, ou ele pode ser medido diretamente em comprimentos de onda do espectro isosbestic extinção molar coeficiente de hemoglobina (por exemplo, 530 nm, 545 nm, 570 nm, e 584 nm) 14, onde HBR e HBO 2 têm iguais coeficientes de extinção molar. 5. Resultados representativos: Mostrado na Figura 1 é a anatomia vascular e sO 2 em uma orelha de rato vivo nua fotografada pela dupla de comprimento de onda (561 e 570 nm) OR-PAM. O tempo de aquisição típicos imagem para comprimento de onda duplo para duas medições de uma região de interesse (tamanho da imagem: 10 mm x 10 mm; tamanho do passo: 2,5 M x 5 mm) é de aproximadamente 80 min. p_upload/2729/2729fig1.jpg &quot;alt =&quot; Figura 1 &quot;/&gt; Figura 1. Em microscopia óptica com resolução vivo fotoacústica. Imagens MAP de (A) a concentração de hemoglobina total mostrando a anatomia vascular (adquirida a 570 nm) e (B) a saturação de oxigênio da hemoglobina (adquirida em 561 nm e 570 nm) em uma orelha do camundongo nude. (C) Close-up da região encaixotado no painel (A). A barra de escala no painel (A) aplica-se tanto (A) e (B).

<ol>
	<li>Maslov, K., Zhang, H. F., Hu, S., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical-resolution+photoacoustic+microscopy+for+in+vivo+imaging+of+single+capillaries.">Optical-resolution photoacoustic microscopy for in vivo imaging of single capillaries.</a> Opt. Lett. 33, 929-931 (2008).</li><li>Zhang, C., Maslov, K., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subwavelength-resolution+label-free+photoacoustic+microscopy+of+optical+absorption+in+vivo.">Subwavelength-resolution label-free photoacoustic microscopy of optical absorption in vivo.</a> Opt. Lett. 35, 3195-3197 (2010).</li><li>Wang, Y., Maslov, K., Kim, C., Hu, S., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+photoacoustic+and+fluorescence+confocal+microscopy.">Integrated photoacoustic and fluorescence confocal microscopy.</a> IEEE. Trans. Biomed. Eng. 57, 2576-2578 (2010).</li><li>Jiao, S., Xie, Z., Zhang, H. F., Puliafito, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+multimodal+imaging+with+integrated+photoacoustic+microscopy+and+optical+coherence+tomography.">Simultaneous multimodal imaging with integrated photoacoustic microscopy and optical coherence tomography.</a> Opt. Lett. 34, 2961-2963 (2009).</li><li>Hu, S., Maslov, K., Tsytsarev, V., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+transcranial+brain+imaging+by+optical-resolution+photoacoustic+microscopy.">Functional transcranial brain imaging by optical-resolution photoacoustic microscopy.</a> J. Biomed. Opt. 14, 040503-040503 (2009).</li><li>Hu, S., Yan, P., Maslov, K., Lee, J. M., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+amyloid+plaques+in+a+transgenic+mouse+model+using+optical-resolution+photoacoustic+microscopy.">Intravital imaging of amyloid plaques in a transgenic mouse model using optical-resolution photoacoustic microscopy.</a> Opt. Lett. 34, 3899-3901 (2009).</li><li>Hu, S., Rao, B., Maslov, K., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+Photoacoustic+Ophthalmic+Angiography.">Label-free Photoacoustic Ophthalmic Angiography.</a> Opt. Lett. 35, 1-3 (2010).</li><li>Jiao, S. L., Jiang, M. S., Hu, J. M., Fawzi, A., Zhou, Q. F., Shung, K. K., Puliafito, C. A., Zhang, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+ophthalmoscopy+for+in+vivo+retinal+imaging.">Photoacoustic ophthalmoscopy for in vivo retinal imaging.</a> Opt. Express. 18, 3967-3972 (2010).</li><li>Oladipupo, S., Hu, S., Santeford, A., Yao, J., Kovalski, J. R., Shohet, R., Maslov, K., Wang, L. V., Arbeit, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+HIF-1+induction+produces+multistage+neovascularization+with+stage-specific+sensitivity+to+VEGFR+inhibitors+and+myeloid+cell+independence.">Conditional HIF-1 induction produces multistage neovascularization with stage-specific sensitivity to VEGFR inhibitors and myeloid cell independence.</a> Blood. , .</li><li>Hu, S., Maslov, K., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+functional+chronic+imaging+of+a+small+animal+model+using+optical-resolution+photoacoustic+microscopy.">In vivo functional chronic imaging of a small animal model using optical-resolution photoacoustic microscopy.</a> Med. Phys. 36, 2320-2323 (2009).</li><li>Hu, S., Maslov, K., Wang, L. 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Biotechnol. 21, 803-806 (2003).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laser Institute of America, American National Standard for Safe Use of Lasers ANSI Z136. , (2007).</li><li>Jacques, S. L., Prahl, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Absorption+of+Hemoglobin+.">Optical Absorption of Hemoglobin .</a> Oregon Medical Laser Center [Internet]. , (1999).</li></ol>

Todos os procedimentos experimentais em animais foram realizados em conformidade com o protocolo de animais de laboratório aprovado pela Escola de Medicina de Animais Estudos Comité de Washington University em St. Louis.

Neste vídeo, nós fornecemos um instruções detalhadas sobre os protocolos experimentais de OR-PAM, incluindo configuração do sistema, alinhamento do sistema, e típica procedimentos experimentais. PAM OR-label-free, não-invasivo tem permitido estudos de funcionamento microvascular e metabolismo em uma base única capilar e, portanto, tem potencial para expandir nossa compreensão da microcirculação relacionados com fisiologia e patologia. Microphotoacoustics já está fabricando este sistema OR-PAM.

Home-made combinador de feixe acústico-ópticos: <ul><li> em ângulo reto prisma (NT32-545, Edmund Optics) </li><li> rombóide prisma (NT49-419, Edmund Optics) </li><li> óleo de silicone (1000cSt, Produtos Clearco) </li><li> OR-PAM sistema (Microphotoacoustics) </li></ul>

three-dimensional optical-resolution photoacoustic microscopy

Microscopia óptica, fornecendo informações valiosas a nível celular e organelas, tem sido amplamente reconhecida como uma tecnologia que permite biomédica. Como os principais pilares da in vivo tridimensional (3-D) microscopia óptica, microscopia de fluorescência single-/multi-photon e tomografia de coerência óptica (OCT) têm demonstrado a sua sensibilidade a contrastes extraordinários de fluorescência e de espalhamento óptico, respectivamente. No entanto, o contraste de absorção óptica dos tecidos biológicos, que codifica a informação fisiológica / patológica essencial, ainda não foi avaliável. O surgimento de photoacoustics biomédica levou a um novo ramo da microscopia óptica, microscopia óptica resolução fotoacústica (OR-PAM) 1, onde a irradiação óptica é voltado para o limite de difração para alcançar um celular ou resolução mesmo nível subcelular duas laterais. Como um complemento valioso para as tecnologias existentes microscopia óptica, OR-PAM traz em pelo menos duas novidades. Primeiro e mais importante, OR-PAM detecta contrastes absorção óptica com extraordinária sensibilidade (ou seja, 100%). Combinando OR-PAM com microscopia de fluorescência com 3 ou óptico-espalhamento baseado 04 de outubro (ou com ambos) fornece abrangente propriedades ópticas dos tecidos biológicos. Segundo, OR-PAM codifica absorção óptica em ondas acústicas, em contraste com os processos de pura óptica em microscopia de fluorescência e outubro, e fornece fundo sem detecção. A detecção acústica em OR-PAM reduz os impactos da dispersão óptica em degradação de sinal e, naturalmente, elimina as interferências possíveis (ou seja, crosstalks) entre a excitação e detecção, que é um problema comum em microscopia de fluorescência devido à sobreposição entre os espectros de excitação e de fluorescência. Único para a imagem latente de absorção óptica, OR-PAM demonstrou ampla aplicações biomédicas desde sua invenção, incluindo, mas não limitado a, neurologia 5, 6, oftalmologia 7, 8, biologia vascular 9 e 10 de dermatologia. Neste vídeo, nós ensinamos a configuração do sistema e alinhamento de OR-PAM, bem como os procedimentos experimentais para a imagem in vivo microvascular funcional.

Watch this Scientific Journal Video about Three-dimensional Optical-resolution Photoacoustic Microscopy at JoVE.com

Three-dimensional Optical-resolution Photoacoustic Microscopy

Óptico com resolução de microscopia fotoacústica (OR-PAM) é uma tecnologia emergente capaz de imagem absorção óptica contrastes<em&gt; In vivo</em&gt; Celulares com resolução e sensibilidade. Aqui, fornecemos uma instrução visualizado em protocolos experimentais de OR-PAM, incluindo o sistema de alinhamento do sistema, configuração, típico<em&gt; In vivo</em&gt; Procedimentos experimentais, e esquemas de imagens funcionais.

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 ...

three-dimensional-optical-resolution-photoacoustic-microscopy

Research

JoVE Journal

Bioengineering

17.7K visualizações.  Washington University in St. Louis. Óptico com resolução de microscopia fotoacústica (OR-PAM) é uma tecnologia emergente capaz de imagem absorção óptica contrastes In vivo Celulares com resolução e sensibilidade. Aqui, fornecemos uma instrução visualizado em protocolos experimentais de OR-PAM, incluindo o sistema de alinhamento do sistema, configuração, típico In vivo Procedimentos experimentais, e esquemas de imagens funcionais.

Vídeo: Three-dimensional Optical-resolution Photoacoustic Microscopy

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Hidrogéis alginato para Tridimensional Cultura Órgão de ovários e tubas uterinas

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Bioengenharia

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Impressão de planar e tridimensional de tintas condutoras

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Photoacoustic Cystography

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The risk of bladder cancers in children due to radiation exposure is more significant than many other cancers. Here we demonstrate the feasibility of nonionizing and noninvasive photoacoustic (PA) imaging of urinary bladders, referred to as photoacoustic cystography (PAC), using near-infrared (NIR) optical absorbents (i.e. methylene blue, plasmonic gold nanostructures, or single walled carbon nanotubes) as an optical-turbid tracer. We have successfully imaged a rat bladder filled with the optical absorbing agents using a dark-field confocal PAC system. After transurethral injection of the contrast agents, the rat's bladders were photoacoustically visualized by achieving significant PA signal enhancement. The accumulation was validated by spectroscopic PA imaging. Further, by using only a laser pulse energy of less than 1 mJ/cm2 (1/20 of the safety limit), our current imaging system could map the methylene-blue-filled-rat-bladder at the depth of beyond 1 cm in biological tissues in vivo. Both in vivo and ex vivo PA imaging results validate that the contrast agents were naturally excreted via urination. Thus, there is no concern regarding long-term toxic agent accumulation, which will facilitate clinical translation.

Photoacoustic cystography (PAC) has a great potential to map urinary bladders, a radiation sensitive internal organ in pediatric patients, without using any ionizing radiation or toxic contrast agent. Here we demonstrate the use of PAC for mapping urinary bladders with an injection of optical-opaque tracers in rats in vivo.

Cistografia fotoacústica

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Cartilagem Human Tissue Fabricação Usando tridimensional Inkjet Tecnologia de Impressão

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

Análise da migração celular dentro de uma matriz de colagénio tridimensional

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

Elaboração e análise de<em&gt; In Vitro</em&gt; Três mama Dimensional Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Engenharia tridimensionais tecidos epiteliais incorporado dentro Matriz Extracelular

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

Fabricação de dispositivos microfluídicos tridimensional baseados em papel para imunoensaios

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the optical mean free path (~1 mm in skin) with high resolution. By combining optical absorption contrast with the high spatial resolution of ultrasound in a single modality, this technique can penetrate deep tissues. Photoacoustic microscopy systems can have either a low acoustic resolution and probe deeply or a high optical resolution and probe shallowly. It is challenging to achieve high spatial resolution and large depth penetration with a single system. This work presents an AR-OR-PAM system capable of both high-resolution imaging at shallow depths and low-resolution deep-tissue imaging of the same sample in vivo. A lateral resolution of 4 &#181;m with 1.4 mm imaging depth using optical focusing and a lateral resolution of 45 &#181;m with 7.8 mm imaging depth using acoustic focusing were successfully demonstrated using the combined system. Here,&#160;in vivo&#160;small-animal blood vasculature imaging is performed to demonstrate its biological imaging capability.

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Here a switchable acoustic resolution (AR) and optical resolution (OR) photoacoustic microscopy (AR-OR-PAM) system capable of both high resolution imaging at shallow depth and low resolution deep tissue imaging on the same sample in vivo is demonstrated.

Microscopia fotoacústica de resolução acústica e óptica comutável para<em&gt; In Vivo</em&gt; Imagem de Vasculatura de Sangue de Pequeno Animal

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.