Queremos agradecer el apoyo del Departamento de Ingeniería Biológica y la Christopher S. Bond Vida del Centro de Ciencias de la Universidad de Missouri. Reconocemos el apoyo beca de Missouri Ciencias de la Vida de Investigación 09-1034 Junta y los NIH R21CA139186-0. También agradecemos a la Ciencias de la Vida cursos del Programa de Oportunidades de Investigación de la Universidad de Missouri, la Universidad de Missouri moleculares básicos de Citología, y la Universidad de Missouri Facultad de Ingeniería de apoyo financiero.

1. Preparación de muestras de sangre <ol><li> Dibuja aproximadamente 10 ml de sangre total de un paciente de cáncer en etapa IV en dos vacutainers 5 ml. </li><li> Centrifugar los tubos durante diez minutos a 3.000 revoluciones por minuto (rpm). </li><li> La muestra de sangre se separa en tres capas diferentes: la capa inferior comprende los eritrocitos, la capa intermedia, llamada también la capa leucocitaria, contiene leucocitos y las células del melanoma, y la capa superior contiene el plasma y las plaquetas. Eliminar el plasma tanto como sea posible sin afectar la capa leucocitaria. </li><li> Colocar 250 ml del Histopaque 1077 en dos tubos de 1 ml Wintrobe. </li><li> El uso de un 9 pipeta &quot;Pasteur, extraer el plasma restante, toda la capa leucocitaria, e incluso la capa superior de los eritrocitos, asegurándose de recoger &lt;500 mL. </li><li> Ponga la solución encima de la capa Histopaque y centrifugar los tubos Wintrobe en 15 ml tubos cónicos de 10 minutos a 3000 rpm. </li><li> La sangre de nuevoseparados, pero esta vez los eritrocitos serán separados por la Histopaque, lo que permite una fácil extracción de la capa leucocitaria. Use un 9 &quot;con una pipeta Pasteur para obtener toda la capa leucocitaria y colocarlo en un tubo de 2 ml eppendorf. </li><li> Suspensión de la capa leucocitaria en 1,5 ml de PBS con un 2% v / v de Tween 20. </li></ol> 2. Flujo de Cámara de Construcción <ol><li> Obtener un cilindro de acrílico, 20 mm de diámetro exterior, diámetro interior 17 mm y 8 mm de alto. Perforar tres agujeros de 5 mm de diámetro a través del lado del cilindro en ángulos de 90 grados uno del otro. Corte tres piezas de tubo de polímero (1,6 mm de diámetro interior, 5 mm de diámetro exterior), y colocarlos en los agujeros perforados. </li><li> Parafilm tramo en la parte inferior del anillo de acrílico para hacer un sello impermeable. La parafina forma un recipiente poco profundo con el anillo de acrílico y llevará a cabo la solución de acrilamida que se forma una cámara de flujo. </li><li> Suspender a un alambre de 1,6 mm de diámetro a través de las piezas de tubería que están al otro lado de laentre sí, asegurándose de que el cable solo es visible a través del centro del ring. Llene la tercera pieza de tubo con un alambre de 1,6 mm de diámetro y la posición del tubo de 1 mm de distancia del cable de suspensión. Estos cables impiden la acrilamida de entrar en el tubo. Después de que la acrilamida se solidifica, los cables se extrae, dejando un camino de flujo cilíndrica y un lugar para colocar una fibra óptica se centra en la trayectoria del flujo. </li><li> Añadir 5 ml de acrilamida preparados [20 g de acrilamida (Sigma Aldrich), 0,7 g de bisacrilamida (Sigma Aldrich), 100 ml de agua destilada] a un vaso pequeño. Medir 0,02 g de persulfato de amonio (Sigma) y agregarlo a la jarra. Mezcle con una barra de agitación durante 3 minutos. Añadir 20 l tetramethylethylenediamene (Fisher Scientific) en el vaso. Rápidamente vierta la mezcla en el anillo de acrílico, relleno de la parte superior. Después de unos segundos, la solución de gel, haciendo un molde de la cámara de flujo. Con cuidado, tire de los cables fuera, con cuidado de no perturbar el gel de acrilamida. El flujo de Chambre ya está listo para su uso. </li></ol> 3. Flujometría fotoacústica set-up <ol><li> Acústicamente un transductor de par fluoruro de polivinilo acústico piezoeléctrico (Panametrics) a una cámara de flujo, utilizando gel de ultrasonidos directamente sobre la trayectoria del flujo. </li><li> El uso de un Bayoneta, Neill-Concelman (BNC) de cable, conectar el transductor a la entrada de un receptor de banda ancha Ritec-640A (filtro de paso bajo: hacia fuera, filtro de paso alto: 1 MHz, llave de entrada Impedancia: 50 Ohms, la amplificación: 35 dB). Conecte la salida del filtro para el osciloscopio (Tektronix TDS 2024B). Para activar el osciloscopio, un fotodiodo (Thorlabs) se encuentra cerca del transductor y se enchufa en el osciloscopio mediante un cable BNC. </li><li> Un conector en T (piezas pequeñas) se une a un brazo de la cámara de flujo. Dos bombas de jeringa (Braintree Científico) están conectados a las otras ramas del conector. Una jeringa contiene aire, mientras que el otro contendrá la suspensión celular preparada en la parte 1. Fijar las tasas de flujo para100 L / minuto. </li><li> Extraer el tubo pequeño de la cámara de flujo hasta una pequeña bolsa de aire de las formas, e insertar un 1 mm de diámetro núcleo de fibra óptica con una apertura numérica 0,37 en esa ranura. Llene la bolsa con agua para reducir las señales de interfaz y aumentar la cantidad de luz del láser aplicado a la trayectoria del flujo. Debido a la apertura numérica de la fibra óptica ya muy poca distancia de la trayectoria del flujo, el láser sólo irradian una babosa de la muestra en un momento dado, asegurando que la muestra directamente en frente del láser es la misma muestra la creación de señales fotoacústica. La energía del láser debe ser de aproximadamente 28 mJ / cm 2, con un tamaño de spot de 7 mm 2. </li><li> A su vez las dos bombas de jeringa. Cuando los dos fluidos inmiscibles llegar al cruce que se dividirá en las babosas homogénea y el flujo más allá del transductor. </li><li> Irradiar la muestra fluye con un nanosegundo láser pulsado para generar ondas de fotoacústica en las células del melanoma. Como la melanina es un broadby ópticos de absorción, cualquier longitud de onda de láser visible puede utilizarse. </li><li> Si aparece una señal en el osciloscopio cuando una bala está en la cima del transductor, la babosa contiene melanoma, y ​​deben ser seguidos a través del sistema hasta caer en un vial de recogida. </li></ol> 4. Inmunocitoquímica <ol><li> Tome las babosas capturadas en una centrífuga Cytospin 4 (Thermo Scientific) con el fin de fijar las diapositivas de cristal. Colocar los portaobjetos de vidrio en Cytofunnels (Shandon Cytofunnel EZ Individual con Tarjeta de Brown filtro Thermo Scientific) y cargar en la Cytospin 4 centrífuga. Ponga 100 L de bovino al 1% de albúmina sérica (Sigma) en PBS en cada embudo y centrifugar a 600 rpm durante 3 minutos para ayudar a las células capturadas se adhieren a las diapositivas de cristal. </li><li> Después de la centrifugación, añadir las muestras de células de la Cytofunnels y luego se centrifuga a 600 rpm durante 3 minutos. Luego rocíe las diapositivas con un fijador de etanol a base. </li><li> Se lavan los portas en PBS durante 10 minutos dos veces. </li><li&gt; para bloquear la unión inespecífica, se incuba cada diapositiva con una mezcla de 10 l de suero de cabra (Sigma) y 90 l de PBS durante 1 hora a temperatura ambiente. </li><li> Después de la incubación, se decanta de las diapositivas y se incuban con la solución de anticuerpo primario durante 1 hora en una cámara húmeda a temperatura ambiente. Cada diapositiva debe contener 1% MART-1 anticuerpo producido en conejo, el 1% de CD-45 anticuerpo en ratones, el 10% de suero de cabra, y el 89% PBS. </li><li> Después de la incubación, lavar los portaobjetos en PBS durante 10 minutos tres veces. </li><li> Las diapositivas se incuban en una solución de anticuerpo secundario, que ha fluoróforo unido a los anticuerpos. La mezcla contiene 0,05% de cabra anti-conejo de anticuerpos, 0,05% de anticuerpo de cabra anti-ratón, el 0,1% de suero de cabra, y el 99% PBS. Los portas se incuban en la oscuridad durante 1 hora a temperatura ambiente. </li><li> Después de la incubación, se decanta de las diapositivas de lavado en PBS durante 5 minutos, tres veces. </li><li> Para la tinción de un núcleo, incubar las células con 0,1 mg / ml de DAPI (Invitrogen) para1 minuto. Entonces decantar las diapositivas y de lavado en PBS. </li><li> Montar un cubreobjetos sobre la muestra de células con 20 L de un medio a base de resina de montaje en cada diapositiva. Sellar el cubreobjetos con esmalte de uñas transparente para preservar la diapositiva. Ahora las diapositivas están listos para ser fotografiado con un microscopio de fluorescencia. </li></ol> 5. Los resultados representativos: <img src="/files/ftp_upload/3559/3559fig1.jpg" alt="Figura 1"/> Figura 1. Las formas de onda fotoacústica de las células blancas de la sangre y las células del melanoma se muestran aquí. Los glóbulos blancos, que no tienen pigmentación inherente, no producen ondas de fotoacústica y se manifiestan como una línea plana de ruido electrónico (izquierda). Las células de melanoma pigmentado produce una onda fuerte fotoacústica (derecha). <img src="/files/ftp_upload/3559/3559fig2.jpg" alt="Figura 2"/> Figura 2. Después de la tinción inmunohistoquímica, las células cultivadas de melanoma muestran señales de DAPI en blue mientras MART1 se resalta en verde. <img src="/files/ftp_upload/3559/3559fig3.jpg" alt="Figura 3"/> Figura 3. Superposición de imágenes para DAPI y MART1, como en la figura 2, con CD45 como un indicador de leucocitos, esta figura muestra las células de melanoma entre varios glóbulos blancos.

<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 es fundador y presidente de Viator Technologies Inc, una compañía formada para comercializar tecnologías fotoacústica para la mejora de la salud humana.

La detección de CTCs es todavía un campo de investigación intensiva con aplicaciones clínicas muy pocos debido al difícil problema de aislar las células del tumor raro. Muchas otras técnicas están siendo evaluados para la detección de CTC, como RT-PCR, la captura de células de microfluidos, la captura inmunomagnética, y otros métodos de 8-12. Sin embargo, la detección fotoacústica de CMC muestra promesa, ya que es la etiqueta libre y proporciona un medio rápido y preciso para capturar las partículas pequeñas, que absorbe la luz. El protocolo descrito para aislar las células blancas de la sangre es muy eficiente, sin embargo, un número bajo de glóbulos rojos existen en la muestra. Estas células rouge contaminantes que absorben la luz láser, pero a un nivel mucho menor. Para asegurarse de que los glóbulos rojos no interfieren con nuestro sistema de detección de melanoma, cinco muestras de pacientes sanos fueron introducidos a través del sistema y no dio una señal, lo que indica que los glóbulos rojos están presentes en bajas concentraciones suficientes paratenerse en cuenta. Una serie de estudios sobre la concentración han llevado a cabo utilizando cultivos de células de melanoma y el sistema de flujo fotoacústica ha demostrado ser sensible a 1 células / l (datos no publicados). Estos estudios están en marcha y pronto incluirá los ensayos con muestras de pacientes con cáncer. Esta técnica fotoacústica está siendo evaluado para su uso en no pigmentados CTC, como el de mama y cáncer de próstata adjuntando cromóforos exógenos. Hemos realizado el trabajo preliminar con nanopartículas de oro sobre el cáncer de cells13. Estas pruebas han demostrado que las nanopartículas pueden proporcionar la absorción óptica necesaria para generar ondas de fotoacústica. El desafío técnico que todavía es unir estas partículas a las células cancerosas de manera selectiva.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </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

Las células tumorales circulantes (CTC) son las células que se han separado de un tumor macroscópico y la difusión a través de los sistemas sanguíneo y linfático a los tumores de semilla secundaria 1,2,3. CTCs son indicadores de la enfermedad metastásica y su detección en muestras de sangre se pueden utilizar para diagnosticar el cáncer y controlar la respuesta del paciente a la terapia. Desde los CTC son raros, que comprende alrededor de una célula tumoral entre los miles de millones de células sanguíneas normales en pacientes con cáncer avanzado, su detección y la enumeración es una tarea difícil. Aprovechamos la presencia del pigmento en la mayoría de las células de melanoma para generar fotoacústica, o inducida por láser las ondas ultrasónicas en un citómetro de flujo a medida para la detección de células de melanoma circulantes (CMC) 4,5. Este proceso implica la separación de una muestra de sangre mediante centrifugación y la obtención de la capa de glóbulos blancos. Si está presente en la sangre, los CMC se separarán con las células blancas de la sangre debido a la densidad similar. Estas células se resuspenden entampón fosfato salino (PBS) y se introduce en el medidor de flujo. En lugar de un flujo continuo de la suspensión de células sanguíneas, que induce el flujo de dos fases con el fin de capturar estas células para estudios posteriores. En el flujo de dos fases, dos líquidos inmiscibles en un sistema de microfluidos se encuentran en un cruce y la forma alterna las babosas de líquido 6,7. PBS suspendidos los glóbulos blancos y las babosas de aire que se forma microlitro de forma secuencial se irradia con luz láser. La adición de un surfactante para la fase líquida permite la formación de bala uniforme y el usuario puede crear diferentes babosas de tamaño mediante la alteración de los caudales de las dos fases. Las babosas de aire y las babosas de PBS con las células blancas de la sangre no contienen absorbentes de luz y por lo tanto, no producen ondas fotoacústica. Sin embargo, los lingotes de glóbulos blancos que contienen incluso CMC solo absorben la luz láser y producir ondas acústicas de alta frecuencia. Estas babosas que generan flujos fotoacústica son secuestradas y se recoge para la tinción citoquímica para verificción de los CMC.

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

Hemos desarrollado un citómetro de flujo con ultrasonido inducida por láser para detectar las células del melanoma circulando como un indicador temprano de la enfermedad metastásica.

La detección y aislamiento de las células de melanoma circulantes mediante Flujometría 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 vistas.  University of Missouri. Hemos desarrollado un citómetro de flujo con ultrasonido inducida por láser para detectar las células del melanoma circulando como un indicador temprano de la enfermedad metastásica.

Video: 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 óptico resolución microscopía fotoacústica

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

Investigación

Bioingeniería

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.

Rápido aislamiento de células tumorales viables de circulación de muestras de sangre del paciente

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.

Bacterial Detección e Identificación Usando sensores electroquímicos

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.

La oftalmoscopia integrado fotoacústica y Spectral-tomografía de coherencia óptica de dominio

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.

Cistografía 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.

Trasplante De InducidaS Pluripotentes Derivadas De Células Madre Mesoangioblastos Como Progenitores Miógenos En Modelos De Ratón De La Regeneración 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.

Individual Detección Molecule-Label gratis con Microtoroid ópticos Resonadores

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.

Análisis preparación y fotoacústica de Vehículos celulares que contienen oro Nanorods

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.

Que circula MicroARN Cuantificación Usando ADN vinculante tinte Química y gotita de PCR digital

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.

Microscopía fotoacústica de resolución óptica y acústica conmutable para<em&gt; In Vivo</em&gt; Imágenes de vasos sanguíneos de pequeños animales

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