Agradecemos o apoio do Departamento de Engenharia Biológica e Christopher S. Life Bond Centro de Ciências da Universidade de Missouri. Reconhecemos apoio financeiro da Missouri Life Sciences Research Board 09-1034 e NIH R21CA139186-0. Também agradecemos ao Programa Life Sciences Research Opportunity Graduação da Universidade de Missouri, a Universidade de Missouri Núcleo Citologia Molecular e da Universidade de Missouri Faculdade de Engenharia de apoio financeiro.

1. Preparação de amostras de sangue <ol><li> Desenhe aproximadamente 10 mL de sangue total de um paciente de cancro Estágio IV em dois tubos sob vácuo 5 mL. </li><li> Centrifugar os tubos durante 10 minutos a 3.000 rotações por minuto (rpm). </li><li> A amostra de sangue será separado em três camadas diferentes: a camada inferior compreende os eritrócitos, a camada intermediária, também chamada de buffy coat, contém leucócitos e células de melanoma, ea camada superior contém plasma e plaquetas. Remover o plasma tanto quanto possível, sem perturbar o buffy coat. </li><li> ML lugar 250 de 1077 Histopaque em dois tubos de um Wintrobe mL. </li><li> Usando um 9 &quot;Pasteur Pipeta, extraia o plasma restante, o revestimento buffy inteiro, e até mesmo a camada superior de eritrócitos, certificando-se de recolher &lt;mL 500. </li><li> Coloque a solução em cima da camada de Histopaque e centrifugar os tubos Wintrobe em 15 mL tubos cônicos por 10 minutos a 3000 rpm. </li><li> O sangue será novamenteseparado, mas desta vez os eritrócitos serão separados pelo Histopaque, permitindo a fácil extração do buffy coat. Use um 9 &quot;Pasteur Pipeta para obter o buffy coat todo e colocá-lo em um tubo eppendorf 2 ml. </li><li> Suspender a buffy coat em 1,5 mL de PBS com 2% v / v de Tween 20. </li></ol> 2. Construção câmara de fluxo <ol><li> Obtenção de um cilindro de acrílico, 20 mm de diâmetro externo, 17 mm de diâmetro interno, e 8 mm de altura. Perfurar três buracos de 5 mm de diâmetro através do lado do cilindro em ângulos de 90 graus um do outro. Corte três pedaços de tubo de polímero (1,6 mm de diâmetro interno, 5 mm de diâmetro externo), e colocá-los nos orifícios perfurados. </li><li> Estiramento Parafilm na parte inferior do anel de acrílico para fazer um selo à prova d&#39;água. O parafilme forma uma bacia rasa com o anel de acrílico e irá realizar a solução de acrilamida que formam uma câmara de fluxo. </li><li> Suspender um fio de 1,6 mm de diâmetro através dos pedaços de tubos que estão em frenteo outro, certificando-se que o fio por si só é visível através do meio do ringue. Preencher a terceira peça de tubo com um fio de diâmetro 1,6 mm e posição do tubo 1 mm de distância do fio suspenso. Estes fios evitar acrilamida de entrar na tubulação. Após a acrilamida se solidifica, os fios serão extraídos, deixando um caminho de fluxo cilíndrico e um lugar para a posição de uma fibra óptica se concentra no caminho de fluxo. </li><li> Adicionar 5 mL de acrilamida preparado [20 acrilamida g (Sigma Aldrich), 0,7 g bisacrylamide (Sigma Aldrich), 100 mL de água destilada] para um copo pequeno. Medir 0,02 g de persulfato de amônio (Sigma) e adicioná-lo no copo. Misture com uma barra de agitação por 3 minutos. Adicionar 20 mL tetramethylethylenediamene (Fisher Scientific) para o copo. Rapidamente despeje a mistura no anel de acrílico, enchendo até o topo. Após alguns segundos, a solução de gel, fazendo um molde de câmara de fluxo. Com cuidado, puxe os fios para fora, tomando cuidado para não perturbar o gel de acrilamida. O fluxo de chamber agora está pronto para usar. </li></ol> 3. Fluxometria fotoacústica set-up <ol><li> Acusticamente um par de polivinilideno de fluoreto transdutor acústico piezoelétrico (Panametrics) para uma câmara de fluxo, utilizando gel de ultra-som diretamente acima do caminho de fluxo. </li><li> Usando uma baioneta Neill-Concelman (BNC) cabo, conectar o transdutor para a entrada de um receptor de banda larga RITEC-640A (filtro passa-baixa: out, highpass filtro: 1 MHz, Impedância de entrada: 50 Ohms, amplificação: 35 dB). Conecte a saída do filtro ao osciloscópio (Tektronix TDS 2024B). Para acionar o osciloscópio, um fotodíodo (Thorlabs), é criada perto do transdutor e é conectado ao osciloscópio através de um cabo BNC. </li><li> A t-connector (Small Parts) é ligado a um braço da câmara de fluxo. Duas bombas de seringa (Braintree Científica) estão ligados a outros ramos do conector. Uma seringa contém ar, enquanto o outro irá conter a suspensão de células preparadas na parte 1. Definir o fluxo para100 L / minuto. </li><li> Extraia o tubo pequeno da câmara de fluxo até que uma pequena bolsa de ar de formas, e inserir um 1 milímetro de diâmetro do núcleo de fibra óptica com uma abertura de 0,37 numérica nesse slot. Encha o bolso com água para reduzir os sinais de interface e aumentar a quantidade de luz laser aplicada ao caminho de fluxo. Devido à abertura numérica da fibra óptica e curta distância para o caminho do fluxo, o laser só vai irradiar um slug de amostra em um dado momento, garantindo que a amostra diretamente na frente do laser é a mesma amostra criando sinais fotoacústica. A energia do laser deve ser cerca de 28 mJ / cm 2, com um tamanho de ponto, de 7 mm 2. </li><li> Vire ambas as bombas de seringa por diante. Quando os dois fluidos imiscíveis chegar ao entroncamento que vai ser particionado em lesmas homogênea e passar ao lado do transdutor. </li><li> Irradiar a amostra flui com um nanossegundo pulsada a laser para gerar ondas fotoacústica nas células de melanoma. Como a melanina é uma broadbe ópticas de absorção, qualquer comprimento de onda do laser visível pode ser usado. </li><li> Se um sinal aparece no osciloscópio quando uma lesma está no topo do transdutor, a lesma contém melanoma, e deve ser monitorado através do sistema até cair em um frasco de coleta. </li></ol> 4. Imunocitoquímica <ol><li> Pegue as lesmas capturado a um 4 Cytospin Centrífuga (Thermo Scientific), a fim de corrigi-los a lâminas de vidro. Colocar as lâminas de vidro em Cytofunnels (Shandon Cytofunnel EZ único com Brown Filtro Cartão Thermo Scientific) e colocá-los na 4 Cytospin centrífuga. Coloque 100 mL de 1% albumina bovina (Sigma) em PBS em cada funil e, em seguida, centrifugar a 600 rpm por 3 minutos para ajudar as células capturadas ater ao lâminas de vidro. </li><li> Após a centrifugação, adicionar as amostras de célula para a Cytofunnels e centrifugar a 600 rpm por 3 minutos. Em seguida, pulverizar a slides com um fixador de etanol à base. </li><li> Lavar as lâminas em PBS por 10 minutos duas vezes. </li><li&gt; Para bloquear a ligação não específica, incubar cada slide com uma mistura de 10 mL de soro de cabra (Sigma) e 90 mL de PBS por 1 hora em temperatura ambiente. </li><li> Após a incubação, decanta os slides e incube com a solução de anticorpo primário por 1 hora em câmara úmida à temperatura ambiente. Cada slide deve conter 1% MART-1 anticorpos produzidos em coelho, um CD-45% de anticorpos criados em mouse, soro de cabra 10%, e PBS 89%. </li><li> Após a incubação, lavar as lâminas em PBS por 10 minutos três vezes. </li><li> As lâminas são incubadas em uma solução de anticorpo secundário, que tem fluoróforo ligado aos anticorpos. A mistura contém 0,05% de cabra anti-coelho de anticorpos, 0,05% de anticorpos de cabra anti-rato, soro de cabra 0,1% e PBS 99%. As lâminas são incubadas no escuro por uma hora em temperatura ambiente. </li><li> Após a incubação, decanta as lâminas de lavagem em PBS por 5 minutos, três vezes. </li><li> A mancha de um núcleo, incubar as células com 0,1 g / mL de DAPI (Invitrogen) para1 minuto. Decantar o slides e lavar em PBS. </li><li> Monte uma lamela sobre a amostra de células usando 20 mL de uma mídia baseada em resina de montagem por slide. Selar a lamela com unhas polonês claro para preservar o slide. Agora os slides estão prontos para ser fotografada com um microscópio de fluorescência. </li></ol> 5. Resultados representativos: <img src="/files/ftp_upload/3559/3559fig1.jpg" alt="Figura 1"/> Figura 1. As formas de onda fotoacústica partir de células brancas do sangue e células de melanoma são mostrados aqui. As células brancas do sangue, não tendo nenhuma pigmentação inerente, não produzem ondas de fotoacústica e se manifestar como uma linha reta de ruídos eletrônicos (à esquerda). As células de melanoma pigmentada produz uma onda robusta fotoacústica (direita). <img src="/files/ftp_upload/3559/3559fig2.jpg" alt="Figura 2"/> Figura 2. Após a coloração imunocitoquímica, células de melanoma cultivadas mostram sinais de DAPI em blue enquanto MART1 é destaque em verde. <img src="/files/ftp_upload/3559/3559fig3.jpg" alt="Figura 3"/> Figura 3. Sobrepondo imagens para DAPI e MART1, como na Figura 2, com CD45 como um indicador de leucócitos, esta figura mostra células de melanoma entre várias células brancas do sangue.

<ol>
	<li>Pantel, K., Brakenhoff, R., Brandt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+clinical+relevance+and+specific+biological+properties+of+disseminating+tumour+cells.">Detection clinical relevance and specific biological properties of disseminating tumour cells.</a> Nature Reviews Cancer. 8, 329-340 (2008).</li><li>Pantel, K., Brakenhoff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+metastatic+cascade.">Dissecting the metastatic cascade.</a> Nature Reviews Cancer. 4, 448-456 (2004).</li><li>Yu, M., Stott, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells:+approaches+to+isolation+and+characterization.">Circulating tumor cells: approaches to isolation and characterization.</a> The Journal of Cell Biology. 192, 373-373 (2011).</li><li>Weight, R. M., Dale, P. S., Viator, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+circulating+melanoma+cells+in+human+blood+using+photoacoustic+flowmetry.">Detection of circulating melanoma cells in human blood using photoacoustic flowmetry.</a> , 106-109 (1985).</li><li>Weight, R. M., Dale, P. S., Lisle, A., Caldwell, C., Viator, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+detection+of+metastatic+melanoma+cells+in+the+human+circulatory+system.">Photoacoustic detection of metastatic melanoma cells in the human circulatory system.</a> Opt. Lett. 31, 2998-3000 (2006).</li><li>Gupta, A., Kumar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+geometry+on+droplet+formation+in+the+squeezing+regime+in+a+microfluidic+T-junction.">Effect of geometry on droplet formation in the squeezing regime in a microfluidic T-junction.</a> Microfluidics and Nanofluidics. 8, 799-812 (2010).</li><li>Mocellin, S., Hoon, D., Ambrosi, A., Nitti, D., Rossi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prognostic+value+of+circulating+tumor+cells+in+patients+with+melanoma:+a+systematic+review+and+meta-analysis.">The prognostic value of circulating tumor cells in patients with melanoma: a systematic review and meta-analysis.</a> Clinical Cancer Research. 12, 4605-4605 (2006).</li><li>Cristofanilli, M., Budd, G., Ellis, M., Stopeck, A., Matera, J., Miller, M., Reuben, J., Doyle, G., Allard, W., Terstappen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells,+disease+progression,+and+survival+in+metastatic+breast+cancer.">Circulating tumor cells, disease progression, and survival in metastatic breast cancer.</a> New England Journal of Medicine. 351, 781-781 (2004).</li><li>Cristofanilli, M., Hayes, D., Budd, G., Ellis, M., Stopeck, A., Reuben, J., Doyle, G., Matera, J., Allard, W., Miller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells:+a+novel+prognostic+factor+for+newly+diagnosed+metastatic+breast+cancer.">Circulating tumor cells: a novel prognostic factor for newly diagnosed metastatic breast cancer.</a> Journal of Clinical Oncology. 23, 1420-1420 (2005).</li><li>Ghossein, R., Scher, H., Gerald, W., Kelly, W., Curley, T., Amsterdam, A., Zhang, Z., Rosai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+circulating+tumor+cells+in+patients+with+localized+and+metastatic+prostatic+carcinoma:+clinical+implications.">Detection of circulating tumor cells in patients with localized and metastatic prostatic carcinoma: clinical implications.</a> Journal of Clinical Oncology. 13, 1195-1195 (1995).</li><li>Loberg, R., Fridman, Y., Pienta, B., Keller, E., McCauley, L., Taichman, R., Pienta, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+isolation+of+circulating+tumor+cells+in+urologic+cancers:+a+review.">Detection and isolation of circulating tumor cells in urologic cancers: a review.</a> Neoplasia. 6, 302-302 (2004).</li><li>Viator, J. A., Gupta, S. K., Goldschmidt, B. S., Bhattacharyya, K., Raghuraman, R., Shukla, R., Dale, P. S., Boote, E., Katti, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+gold+nanoparticle+enhanced+prostate+cancer+cells+using+photoacoustic+flowmetry+with+optical+reflectance.">Detection of gold nanoparticle enhanced prostate cancer cells using photoacoustic flowmetry with optical reflectance.</a> J. Biomed. Nanotech. 6, 187-191 (2010).</li></ol>

John A. Viator é fundador e presidente da Viator Technologies Inc, uma empresa criada para comercializar tecnologias fotoacústica para a melhoria da saúde humana.

Detecção de CTCs ainda é um campo de pesquisa intensiva com muito poucas aplicações clínicas devido ao problema difícil de isolar as células do tumor raro. Muitas outras técnicas estão sendo avaliadas para CTC detecção, incluindo RT-PCR, captura célula microfluídica, captura imunomagnética, e outros métodos 8-12. No entanto, a detecção fotoacústica da mostra CMCs promessa como é rótulo livre e fornece um meio rápido e preciso para capturar pequenas partículas de luz absorvente. O protocolo descrito para isolar as células brancas do sangue é altamente eficiente, mas um baixo número de glóbulos vermelhos existem na amostra. Estas células rouge contaminando também absorvem a luz laser, mas a um nível bastante reduzido. Para garantir os glóbulos vermelhos não interferem com o nosso sistema de detecção de melanoma, cinco amostras de pacientes saudáveis ​​foram introduzidas através do sistema e não deu um sinal, indicando que as células vermelhas do sangue estão presentes em baixas concentrações o suficiente paraser desconsiderada. Uma série de estudos de concentração foram realizados com células de melanoma cultivadas eo sistema de fluxo fotoacústica mostrou sensível até 1 célula / mL (dados não publicados). Estes estudos estão em curso e em breve incluirá ensaios utilizando amostras de doentes de câncer. Esta técnica fotoacústica também está sendo avaliado para uso em não-pigmentadas CTCs, como de mama e câncer de próstata, anexando cromóforos exógenos. Temos realizado um trabalho preliminar usando nanopartículas de ouro sobre o câncer cells13. Estes testes mostraram que as nanopartículas podem proporcionar a absorção óptica necessária para gerar ondas fotoacústica. O desafio técnico é demais para anexar tais partículas de células de câncer seletivamente.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> ABCD1234 </td><td></td></tr></tbody></table>

detection and isolation of circulating melanoma cells using photoacoustic flowmetry

Células tumorais circulantes (CTCs) são as células que se separaram de um tumor macroscópico e difundir através dos sistemas de sangue e da linfa aos tumores secundários de sementes 1,2,3. CTCs são indicadores de doença metastática e sua detecção em amostras de sangue podem ser usados ​​para diagnosticar o câncer e monitorar a resposta do paciente à terapia. Desde CTCs são raros, compreendendo cerca de uma célula tumoral entre bilhões de células sanguíneas normais em pacientes com câncer avançado, a sua detecção e enumeração é uma tarefa difícil. Nós explorar a presença de pigmento na maioria das células do melanoma para gerar fotoacústica, ou laser induzida ondas ultra-sônicas em um citômetro de fluxo personalizado para a detecção de células de melanoma circulantes (CMCs) 4,5. Este processo implica separar uma amostra de sangue total usando centrifugação e obter a camada de células brancas do sangue. Se presente no sangue total, CMCs vai separar com as células brancas do sangue, devido à densidade semelhante. Estas células são ressuspendidas emtampão fosfato (PBS) e introduzido o medidor de vazão. Ao invés de um fluxo contínuo de suspensão de células do sangue, que induziu duas fluxo da fase a fim de capturar essas células para posterior estudo. Em dois fluxo de fase, dois líquidos imiscíveis em um sistema microfluídicos se encontram em uma junção e forma alternada slugs de 6,7 líquidos. PBS suspensão glóbulos brancos e lesmas ar microlitro forma que são seqüencialmente irradiado com luz laser. A adição de um surfactante para a fase líquida permite a formação slug uniforme e que o usuário pode criar diferentes lesmas porte, alterando as taxas de fluxo de duas fases. Lesmas do ar e lesmas de PBS com células brancas do sangue não contém absorvedores de luz e, portanto, não produzem ondas fotoacústica. No entanto, lesmas de células brancas do sangue que contêm mesmo CMCs única absorvem a luz laser e produzir ondas acústicas de alta freqüência. Essas lesmas que geram ondas fotoacústica são seqüestrados e recolhidos para a coloração citoquímica para verificção de CMCs.

Watch this Scientific Journal Video about Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry at JoVE.com

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Nós desenvolvemos um citômetro de fluxo, utilizando ultra-som para detectar a laser induzida circulando células de melanoma como um indicador precoce de doença metastática.

Detecção e isolamento de células de melanoma circulantes usando Fluxometria Fotoacústica

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

detection-isolation-circulating-melanoma-cells-using-photoacoustic

Research

JoVE Journal

Bioengineering

15.7K visualizações.  University of Missouri. Nós desenvolvemos um citômetro de fluxo, utilizando ultra-som para detectar a laser induzida circulando células de melanoma como um indicador precoce de doença metastática.

Vídeo: Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

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

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of &gt;50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

Isolamento rápido de células tumorais viáveis ​​circulantes de amostras de sangue de doentes

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Bacteriana Detecção e Identificação Utilizando sensores eletroquímicos

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging technologies. Existing retinal imaging modalities, such as fundus photography1, confocal scanning laser ophthalmoscopy (cSLO)2, and optical coherence tomography (OCT)3, have significant contributions in monitoring disease onsets and progressions, and developing new therapeutic strategies. However, they predominantly rely on the back-reflected photons from the retina. As a consequence, the optical absorption properties of the retina, which are usually strongly associated with retinal pathophysiology status, are inaccessible by the traditional imaging technologies.
Photoacoustic ophthalmoscopy (PAOM) is an emerging retinal imaging modality that permits the detection of the optical absorption contrasts in the eye with a high sensitivity4-7 . In PAOM nanosecond laser pulses are delivered through the pupil and scanned across the posterior eye to induce photoacoustic (PA) signals, which are detected by an unfocused ultrasonic transducer attached to the eyelid. Because of the strong optical absorption of hemoglobin and melanin, PAOM is capable of non-invasively imaging the retinal and choroidal vasculatures, and the retinal pigment epithelium (RPE) melanin at high contrasts 6,7. More importantly, based on the well-developed spectroscopic photoacoustic imaging5,8 , PAOM has the potential to map the hemoglobin oxygen saturation in retinal vessels, which can be critical in studying the physiology and pathology of several blinding diseases 9 such as diabetic retinopathy and neovascular age-related macular degeneration.
Moreover, being the only existing optical-absorption-based ophthalmic imaging modality, PAOM can be integrated with well-established clinical ophthalmic imaging techniques to achieve more comprehensive anatomic and functional evaluations of the eye based on multiple optical contrasts6,10 . In this work, we integrate PAOM and spectral-domain OCT (SD-OCT) for simultaneously in vivo retinal imaging of rat, where both optical absorption and scattering properties of the retina are revealed. The system configuration, system alignment and imaging acquisition are presented.

Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.

Oftalmoscopia Fotoacústica integrada e Spectral-domínio Tomografia de Coerência Óptica

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging ...

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 ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Transplante de Progenitores Miogênicos derivados de células-tronco pluripotentes induzidos em modelos de regeneração muscular

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

Único Detecção de Moléculas livre-Label Usando Microtoroid ópticos Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

Preparação e Fotoacústica Análise de Veículos celulares contendo ouro nanobastões

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

Circulando MicroRNA Quantificação Usando DNA de ligação Dye Química e Gota Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

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 ...