The authors thank Prof. Ralph Weissleder, Dr. Pat Zanzonico, and Dr. NagaVaraKishore Pillarsetty for helpful conversations and the NIH for funding (BMZ: 1K99CA178205-01A1)

ÉTICA DECLARACIÓN: Todos los experimentos con animales en vivo descritos fueron realizados de acuerdo con un protocolo aprobado y bajo las directrices éticas del Memorial Sloan Kettering Cancer Center Institucional Cuidado de Animales y el empleo Comisión (IACUC). 1. Síntesis de Tz-Bn-NOTA <ol><li> En un pequeño recipiente de reacción, se disuelven 7 mg NH 2--Bn NOTA (1,25 x 10 -2 mmol) en 600 l de NaHCO3 tampón (0,1 M, pH 8,1). Comprobar el pH de la solución. Si es necesario, ajustar el pH de la solución a 8,1 usando pequeñas alícuotas de 0,1 M Na 2 CO 3. </li><li> Añadir la solución de NH 2--Bn NOTA a 0,5 mg Tz-NHS (1,25 x 10 -3 mmol) en un tubo de microcentrífuga de 1,7 ml. NOTA: El Tz-NHS o bien se puede pesar a cabo en seco o se añade de una solución madre de DMF seco o DMSO (&lt;50 l). </li><li> Dejar que la solución de reacción resultante reaccionara durante 30 min a RTcon agitación suave. </li><li> Después de 30 min, purificar el producto mediante cromatografía de C 18 HPLC de fase inversa para eliminar sin reaccionar NH 2--Bn NOTA. El NH 2 -Bn-NOTA puede ser monitoreado en una longitud de onda de 254 nm, mientras que el Tz-NHS y Tz-Bn-NOTA están mejor controlados a una longitud de onda de 525 nm. NOTA: Los tiempos de retención son, evidentemente, muy dependiente de la configuración del equipo de HPLC de cada laboratorio (bombas, columnas, tubos, etc.). Sin embargo, para presentar un ejemplo, si un gradiente de 0: 100 MeCN / H 2 O (ambos con 0,1% TFA) a 100: 0 MeCN / H 2 O durante 25 min y una columna analítica 4,6 x 250 mm C 18 se utiliza , los tiempos de retención de Tz-Bn-NOTA, Tz-NHS, y NH-2 -Bn NOTA será de alrededor de 15 min, 16,5 min y 10 min, respectivamente. El producto puede ser purificado de los otros componentes de la reacción, ya sea en una sola carrera o carreras múltiples utilizando una columna de HPLC C 18 semi-preparativa o preparativa. 1 H-NMR, analytical HPLC y ESI-MS son todos los métodos que se pueden utilizar para verificar la pureza del precursor Tz-Bn-NOTA completado. 11 </li><li> Congelar el eluyente HPLC recogido utilizando nitrógeno líquido. </li><li> Envolver el tubo de recogida de congelados en papel de aluminio opaca. </li><li> Colocar el tubo de recogida congelado en un recipiente de liofilización de O / N para eliminar la fase móvil de HPLC. </li><li> Almacenar el producto purificado (un rosa brillante sólido) en la oscuridad a -80 ° C. NOTA: Este es un punto de parada aceptable en el procedimiento. El precursor Tz-Bn-NOTA completado es estable durante al menos 1 año en estas condiciones. </li></ol> 2. Preparación de huA33-TCO Inmunoconjugado <ol><li> En un tubo de microcentrífuga de 1,7 ml, preparar una 1 mg / ml (2,7 mM) de solución TCO-NHS en DMF seca. </li><li> En un tubo de microcentrífuga de 1,7 ml, preparar una 2 mg / ml (13,3 mM) de huA33 en 1 ml de tampón fosfato salino, pH 7,4 (0,01 M PO 4 3-, </sub&gt; 0,154 M NaCl). </li><li> El uso de pequeñas alícuotas (&lt;5 mu l) de 0,1 M Na 2 CO 3, ajustar el pH de la solución de anticuerpo a 8,8-9,0. Utilice papel de pH o un medidor de pH con un microelectrodo para monitorear el pH, y tener cuidado de no permitir que el pH se eleve por encima de pH 9,0. </li><li> Una vez que la solución de anticuerpo está en el pH correcto, añadir un volumen de la solución TCO-NHS correspondiente a 8 equivalentes molares del éster activado. Por ejemplo, añadir 7,9 l de la solución de 1 mg / ml TCO-NHS (1,07 x 10 -7 mol TCO-NHS) a los 1 ml de solución de 2 mg / ml de anticuerpo huA33 (1,33 x 10 -8 mol huA33). No superar el 5% de DMF en volumen en la solución. </li><li> Mezclar suavemente la solución invirtiendo el tubo de microcentrífuga varias veces. </li><li> Envuelva el tubo de microcentrífuga en papel de aluminio opaco. </li><li> Deje que la solución se incuba durante 1 hora a temperatura ambiente con agitación suave. </li><li> Después de 1 hora a temperatura ambiente, purificar el inmunoconjugado resultante utilizando un siz desechable preenvasadose exclusión columna de desalación. En primer lugar, enjuagar la columna de exclusión de tamaño como se describe por el proveedor para eliminar cualquier conservantes presentes en la columna durante el almacenamiento. A continuación, añadir la mezcla de reacción a la columna de exclusión por tamaño, enjuagar la columna con 1.5 ml 0.9% de solución salina estéril, y posteriormente recoger el producto usando 2 ml de 0,9% de solución salina estéril como eluyente. NOTA: Este paso será dar el terminado huA33-TCO como una solución de 2 ml. </li><li> Medir la concentración de la resultante huA33-TCO en un espectrofotómetro UV-Vis. </li><li> Si se desea una mayor concentración, concentrar la solución huA33-TCO utilizando una unidad de filtro centrífugo con un peso molecular 50.000 de corte. NOTA: Es importante observar que mientras que huA33 y una variedad de otros anticuerpos conocidos (por ejemplo, bevacizumab, trastuzumab, cetuximab, y J591) son muy tolerantes de concentrarse, la agregación y la precipitación pueden ocurrir después de la concentración en otros casos. Los investigadores de intentar este procedUre con un nuevo anticuerpo debe confiar en la literatura o su propio conocimiento del anticuerpo en cuestión con respecto a si es o no concentrar el anticuerpo. </li><li> Guarde el inmunoconjugado huA33-TCO completado a 4 ° C en la oscuridad. NOTA: Este es un punto de parada aceptable en el procedimiento. El conjugado de mAb-TCO completado debe ser estable durante al menos 3 meses en estas condiciones de almacenamiento. </li></ol> 3. 64 Cu radiomarcaje de Tz-Bn-NOTA NOTA: Este paso del protocolo implica el manejo y manipulación de la radiactividad. Antes de realizar estos pasos - o la realización de cualquier otro trabajo con radiactividad - investigadores deben consultar con el Departamento de Seguridad Radiológica de su institución de origen. Tomar todas las medidas posibles para minimizar la exposición a la radiación ionizante. <ol><li> En un tubo de microcentrífuga de 1,7 ml, preparar una 0,5 mg / ml (723 mM) de solución de Tz-Bn-NOTA. </li><li> En una MicroC 1,7 mltubo entrifuge, añadir 10 l de la solución Tz-Bn-NOTA (5 mg) a 400 l de NH4OAc 0,2 M, pH 5,5. </li><li> En aras de la radioquímica nota de mantenimiento de la correcta, medir y registrar la cantidad de radiactividad en la muestra usando un calibrador de dosis antes y después de los pasos siguientes en el protocolo a continuación (3.4 a 3.8). Esto le ayudará con la determinación precisa de los rendimientos de radioquímica. </li><li> Agregar 2000 Ci (74 MBq) de 64 Cu a la solución Tz-Bn-NOTA. NOTA: Típicamente, [64 Cu] CuCl 2 se suministra en un volumen pequeño (&lt;30 l) de HCl 0,1 N, y por tanto sólo pequeños volúmenes (&lt;10 l) de esta solución madre se necesitan para la reacción de radiomarcado. Si se necesitan mayores volúmenes de la [64 Cu] CuCl 2 stock, la reacción de radiomarcaje es tolerante de aumentar el volumen de reacción total. Sin embargo, el pH de la solución de reacción de radiomarcaje debe controlarse cuidadosamente para asegurarque no caiga por debajo de pH 4,0. </li><li> Deje que la solución se incuba durante 10 min a TA con agitación suave. </li><li> Después de 10 min de incubación, purificar el producto mediante cromatografía de HPLC de fase inversa C 18. Los tiempos de retención son, evidentemente, muy dependiente de la configuración del equipo de HPLC de cada laboratorio (bombas, columnas, tubos, etc.). Sin embargo, para presentar un ejemplo, si un gradiente de 5:95 MeCN / H 2 O (ambos con 0,1% TFA) a 95: 5 MeCN / H 2 O durante 15 min se utiliza, el tiempo de retención de 64 Cu-Tz- Bn-NOTA debe estar alrededor de 9,8 minutos, mientras gratis, no acomplejada 64 Cu eluirá con el frente del disolvente en torno a 2-4 min. </li><li> Usando un evaporador rotatorio, retire el eluyente HPLC. </li><li> Se vuelve a disolver el producto 64 Cu-Tz-Bn-nota en el 0,9% de solución salina estéril. NOTA: Dado el 12,7 hr vida media física de 64 Cu, esto no es un punto de parada aceptable en el procedimiento. Realizar la síntesis de 64 Cu-Tz-Bn-NOA inmediatamente antes de la inyección del radioligando, y seguir paso a paso inmediatamente 3,7 4,5. </li></ol> 4. En Vivo Pretargeted TEP NOTA: Como en la sección Protocolo 3, este paso del protocolo implica el manejo y manipulación de la radiactividad. Antes de realizar estos pasos investigadores deben consultar con el Departamento de Seguridad Radiológica de su institución de origen. Tomar todas las medidas posibles para minimizar la exposición a la radiación ionizante. <ol><li> En un ratón atímicos hembra, implante subcutáneamente 1 x 10 células de cáncer colorrectal 6 SW1222 y permita que éstas se conviertan en un xenotrasplante 100-150 mm 3 (9-12 días después de la inoculación). 11 </li><li> Diluir una alícuota de la solución huA33-TCO de la sección Protocolo 2 a una concentración de 0,5 mg / ml en 0,9% de solución salina estéril. </li><li> Inyectar 200 l de la solución huA33-TCO (100 g) en la vena de la cola del ratón de xenoinjerto de soporte. </li><li> Permita 24 horas para la acumulación de la huA33-TCO en el tumor del ratón. </li><li> Diluir el 64 Cu-Tz-Bn-NOTA radioligando a una concentración de 1,5 mCi / ml en 0,9% de solución salina estéril. </li><li> Inyectar 200 l de la solución de radioligando Cu-Tz-Bn-NOTA 64 (300 Ci; 11,1 MBq; 1,6 nmol de 64 Cu-Tz-Bn-NOTA, asumiendo una actividad específica de 6,7 MBq / nmol) en la vena de la cola de la xenoinjerto ratones portadores. </li><li> En el punto de tiempo de formación de imágenes deseado (por ejemplo, 2, 6, 12, o 24 horas después de la inyección), anestesiar al ratón con un isoflurano 2%: mezcla de gas de oxígeno. </li><li> Coloque el ratón sobre la cama del pequeño escáner PET animal. Mantener la anestesia durante el análisis utilizando un isoflurano al 1%: mezcla de gas de oxígeno. Antes de colocar el animal en el escáner, verifique la anestesia utilizando el método del dedo del pie-pinch y aplicar ungüento veterinario a los ojos del ratón para evitar que se seque durante la anestesia. </li><li> Adquirir los datos de PET para el ratón a través de una estáticaescanear con un mínimo de 20 millones de eventos coincidentes utilizando una ventana de energía de 350-700 keV y una ventana de tiempo coincidencia de 6 nseg. Después de completar la adquisición de la imagen, no deje el ratón sin atención y no lo coloque en una jaula con otros ratones hasta que se haya recuperado la conciencia. </li></ol>

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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A33+antigen+displays+persistent+surface+expression.">A33 antigen displays persistent surface expression.</a> Cancer Immunology and Immunotherapy. 57, 1017-1027 (2008).</li><li>Carrasquillo, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=124I-huA33+antibody+PET+of+colorectal+cancer.">124I-huA33 antibody PET of colorectal cancer.</a> Journal of Nuclear Medicine. 52, 1173-1180 (2011).</li><li>Sakamoto, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+I+radioimmunolocalization+trial+of+humanized+monoclonal+antibody+huA33+in+patients+with+gastric+carcinoma.">A phase I radioimmunolocalization trial of humanized monoclonal antibody huA33 in patients with gastric carcinoma.</a> Cancer Science. 97, 1248-1254 (2006).</li><li>Rossin, R., Lappchen, R., vanden Bosch, S. M., LaForest, R., Robillard, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diels-Alder+reaction+for+tumor+pretargeting:+In+vivo+chemistry+can+boost+tumor+radiation+dose+compared+with+directly+labeled+antibody.">Diels-Alder reaction for tumor pretargeting: In vivo chemistry can boost tumor radiation dose compared with directly labeled antibody.</a> Journal of Nuclear Medicine. 54, 1989-1995 (2013).</li><li>Rossin, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+reactive+trans-cyclooctene+tags+with+improved+stability+for+Diels-Alder+chemistry+in+living+systems.">Highly reactive trans-cyclooctene tags with improved stability for Diels-Alder chemistry in living systems.</a> Bioconjugate Chemistry. 34, 1210-1217 (2014).</li><li>Emmetiere, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=18F-labeled-bioorthogonal+liposomes+for+in+vivo+targeting.">18F-labeled-bioorthogonal liposomes for in vivo targeting.</a> Bioconjugate Chemistry. 24, 1784-1789 (2013).</li></ol>

The authors have nothing to disclose.

La principal ventaja de esta estrategia de formación de imágenes PET pretargeted es que es capaz de delinear los tumores con contraste de la imagen-objetivo a fondo a sólo una fracción de la dosis de radiación de fondo producida por los anticuerpos marcados directamente. Por ejemplo, en el sistema de imágenes de cáncer colorrectal se describe aquí, se emplearon datos de los experimentos de biodistribución agudos para realizar cálculos de dosimetría para la estrategia de predireccionamiento a base de Cu 6...

Durante los últimos treinta años, la tomografía por emisión de positrones (PET) se ha convertido en una herramienta clínica indispensable en el diagnóstico y tratamiento del cáncer. Los anticuerpos han sido considerados vectores prometedores para la entrega de radioisótopos emisores de positrones a los tumores debido a su exquisita afinidad y especificidad para biomarcadores de cáncer. 1,2 farmacocinética Sin embargo, la relativamente lentos in vivo de anticuerpos ordena el uso de radioisótopos con multi-día semividas físicas. Esta combinación puede producir altas dosis de radiación a los órganos que no son objeto de los pacientes, una complicación importante que es de particular importancia clínica ya radioinmunoconjugados se inyectaron por vía intravenosa, por lo que - a diferencia de las tomografías computarizadas cuerpo parciales - resultado en dosis absorbidas en cada parte del cuerpo, independientemente de los tejidos interrogados. Para evitar este problema, esfuerzo significativo se ha dedicado a la desallo de estrategias de formación de imágenes de PET que desacoplar el radioisótopo y el resto de dirección, aprovechando así las propiedades ventajosas de anticuerpos bordeando simultáneamente sus limitaciones intrínsecas farmacocinéticos. Estas estrategias - más a menudo denominados predireccionamiento o focalización de varios pasos - emplean típicamente cuatro pasos: (1) la administración de un anticuerpo capaz de unirse tanto un antígeno y un radioligando; (2) la acumulación del anticuerpo en el tejido diana y su aclaramiento de la sangre; (3) la administración de una pequeña molécula de radioligando; y (4) la ligación in vivo del radioligando con el anticuerpo seguido por la rápida eliminación del exceso de radioligando. 3-8 En algunos casos, se inyecta un agente de compensación adicional entre los pasos 2 y 3 con el fin de acelerar la excreción de cualquier anticuerpo Sin embargo, eso tiene que obligar a la tumor y permanece en la sangre. 5 En términos generales, twtipos o de estrategias predireccionamiento son más frecuentes en la literatura. Mientras que ambos han demostrado su eficacia en modelos preclínicos, también poseen limitaciones clave que han impedido su aplicabilidad clínica. La primera estrategia se basa en la alta afinidad entre los anticuerpos conjugados con estreptavidina y radiomarcadores modificados con biotina; Sin embargo, la inmunogenicidad de los anticuerpos modificados con estreptavidina ha demostrado ser un problema preocupante con respecto a la traducción. 5,6,9,10 La segunda estrategia, en cambio, emplea anticuerpos biespecíficos que han sido genéticamente modificados para-unirse tanto un cáncer antígeno biomarcador y un pequeño hapteno marcado radiactivamente molécula. 3,11-14 Mientras que esta última ruta es ciertamente creativo, su amplia aplicabilidad está limitada por la complejidad, gasto, y la falta de modularidad del sistema. Recientemente, hemos desarrollado y publicado una metodología de formación de imágenes PET pretargeted basado en la demanda de electrones inversa Diels-Alder (IEdda) reacción de cicloadición entre -cyclooctene trans (TCO) y tetrazina (Tz;. Figura 1) 11 Mientras que la reacción misma se conoce desde hace décadas, la química IEDDA ha experimentado un renacimiento en los últimos años como una técnica bioconjugación de química, como se ilustra en el fascinante trabajo de los grupos de Ralph Weissleder, Joseph Fox, y Peter Conti entre otros. 12.15 El IEDDA cicloadición se ha aplicado en una amplia gama de entornos, incluyendo imágenes de fluorescencia con péptidos, anticuerpos, y nanopartículas, así como imágenes nucleares . con ambas radiohalogens y radiometales 16-26 La ligadura es de alto rendimiento, limpia, rápida (k 1&gt; 30000 M -1 s -1), selectiva y - crítica -. bioorthogonal 27 Y mientras que una serie de tipos de química modular - incluyendo cicloadiciones Cu-catalizada por azida alquino, cicloadiciones-azida alquino deformación-promovido y Staudinger ligciones -. bioorthogonal son, así, es la combinación única de la cinética de reacción rápidos y bioorthogonality que hace que la química IEDDA tan bien adecuado para el predireccionamiento aplicaciones en organismos enteros 28,29 A lo largo de estas líneas, es importante tener en cuenta que el reciente informe de nuestro laboratorios no fue el primero en aplicar la química IEDDA a predireccionamiento: el primer informe de la imagenología pretargeted con IEDDA surgió del trabajo de Rossin, y otros, y contó con una metodología que emplea un SPECT 111 tetrazina En marcado 30.. Como ya comentamos anteriormente, la metodología predireccionamiento tiene cuatro pasos muy simples (Figura 2). En el protocolo a la mano, se describirá una estrategia pretargeted para la formación de imágenes PET de cáncer colorrectal que se emplea un 64 marcado con Cu-NOTA radioligando tetrazina y un conjugado modificado-TCO del anticuerpo huA33. Sin embargo, en última instancia, la modularidad de esta metodología es uno de sus grcomieres activos, como el resto -cyclooctene trans se pueden adjuntar a cualquier anticuerpo no internalización, y la tetrazina se pueden unir a una amplia variedad de reporteros radiactivos.

<table border="0" cellpadding="0" cellspacing="0" width="565">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;">Tetrazine NHS Ester</td>
			<td>Sigma-Aldrich</td>
			<td>764701</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trans-cyclooctene NHS Ester</td>
			<td>Sigma-Aldrich</td>
			<td>764523</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">p-NH2-Bn-NOTA</td>
			<td>Macrocyclics</td>
			<td>B-601</td>
			<td>Store at -80 &#176;C</td>
		</tr>
	</tbody>
</table>

harnessing the bioorthogonal inverse electron demand diels-alder cycloaddition for pretargeted pet imaging

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for PET imaging. However, the necessity of labeling antibodies with radionuclides with long physical half-lives often results in high background radiation dose rates to non-target tissues. In order to circumvent this issue, we have employed a pretargeted PET imaging strategy based on the inverse electron demand Diels-Alder cycloaddition reaction. The methodology decouples the antibody from the radioactivity and thus exploits the positive characteristics of antibodies, while eschewing their pharmacokinetic drawbacks. The system is composed of four steps: (1) the injection of a mAb-trans-cyclooctene (TCO) conjugate; (2) a localization time period during which the antibody accumulates in the tumor and clears from the blood; (3) the injection of the radiolabeled tetrazine; and (4) the in vivo click ligation of the components followed by the clearance of excess radioligand. In the example presented in the work at hand, a 64Cu-NOTA-labeled tetrazine radioligand and a trans-cyclooctene-conjugated humanized antibody (huA33) were successfully used to delineate SW1222 colorectal cancer tumors with high tumor-to-background contrast. Further, the pretargeting methodology produces high quality images at only a fraction of the radiation dose to non-target tissue created by radioimmunoconjugates directly labeled with 64Cu or 89Zr. Ultimately, the modularity of this protocol is one of its greatest assets, as the trans-cyclooctene moiety can be appended to any non-internalizing antibody, and the tetrazine can be attached to a wide variety of radioisotopes.

Los tres pasos iniciales del experimento - la síntesis de Tz-Bn-NOTA, la conjugación de TCO para huA33, y el radiomarcado del Tz-Bn-NOTA construyen (Figuras 3 y 4) - son altamente confiables. En el caso del procedimiento anterior, el constructo Tz-Bn-NOTA se sintetizó de alto rendimiento y pureza. El anticuerpo huA33 fue modificado con 4,2 ± 0,6 TCO / mAb, y Tz-Bn-NOTA fue radiomarcado con 64 Cu para producir el radioligando purificada en&gt; 99% de pureza radioquímica,&...

Watch this Scientific Journal Video about Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging at JoVE.com

Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand.

El aprovechamiento de la demanda Bioorthogonal Inverse Electron Diels-Alder Cicloadición para Pretargeted TEP

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for ...

harnessing-bioorthogonal-inverse-electron-demand-diels-alder

Research

JoVE Journal

Bioengineering

8.7K vistas.  Memorial Sloan Kettering Cancer Center. The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand. 

Video: Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

De alta resolución de fibra óptica para Microendoscopy In situ Celular imágenes

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Investigación

Bioingeniería

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans&#039; Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

Electrotaxis microfluidos basados ​​en On-Demand Análisis cuantitativo de<em&gt; Caenorhabditis elegans</em&gt; &#39;Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Creación de degradados adhesivas y soluble para la migración celular con imágenes de microscopía de fluorescencia

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Fluorescencia de extinción de un fluoróforo encapsulada en liposomas en infrarrojo cercano como herramienta para<em&gt; En Vivo</em&gt; Imagen Óptico

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

El rápido escaneo de electrones resonancia paramagnética abre nuevas vías para Imaging fisiológicamente parámetros importantes<em&gt; En Vivo</em

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

Correlativos luz microscopia electrónica (CLEM) para el seguimiento y proyección de imagen proteína Viral asociada a estructuras en las células inmovilizadas de Cryo

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various factors affect the relaxation rates, such as buffer composition, solution pH, temperature, and magnetic field. In this last regard, the spin-lattice relaxation time can be measured at clinical field strengths, but at lower fields, where these compounds are dispensed from the polarizer and transported to the MRI, the relaxation is even faster and difficult to measure. To have a better understanding of the amount of magnetization lost during transport, we used fast field-cycling relaxometry, with magnetic resonance detection of 13C nuclei at ~0.75 T, to measure the nuclear magnetic resonance dispersion of the spin-lattice relaxation time of hyperpolarized [1-13C]pyruvate. Dissolution dynamic nuclear polarization was used to produce hyperpolarized samples of pyruvate at a concentration of 80 mmol/L and physiological pH (~7.8). These solutions were rapidly transferred to a fast field-cycling relaxometer so that relaxation of the sample magnetization could be measured as a function of time using a calibrated small flip angle (3&#176;-5&#176;). To map the T1 dispersion of the C-1 of pyruvate, we recorded data for different relaxation fields ranging between 0.237 mT and 0.705 T. With this information, we determined an empirical equation to estimate the spin-lattice relaxation of the hyperpolarized substrate within the mentioned range of magnetic fields. These results can be used to predict the amount of magnetization lost during transport and to improve experimental designs to minimize signal loss.

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

We present a protocol to measure the magnetic field dependence of the spin-lattice relaxation time of 13C-enriched compounds, hyperpolarized by means of dynamic nuclear polarization, using fast field-cycled relaxometry. Specifically, we have demonstrated this with [1-13C]pyruvate, but the protocol could be extended to other hyperpolarized substrates.

Medición de la dependencia del campo magnético de relajación Spin-Lattice de hiperpolarizado [1-13C]pyruvate

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various ...

Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built high viscous injector, Lipidico, as part of the macromolecular crystallography (MX2) beamline to measure large numbers of small crystals at room temperature. The goal of this technique is to enable crystals to be grown/transferred to&#160;glass syringes to be used directly in the injector for serial crystallography data collection. The advantages of this injector include the ability to respond rapidly&#160;to changes in the flow rate without interruption of the stream. Several limitations for this high viscosity injector (HVI) exist which include a restriction on the allowed sample viscosities to &#62;10 Pa.s. Stream stability can also potentially be an issue depending on the specific properties of the sample. A detailed protocol for how to set up samples and operate the injector for serial crystallography measurements at the Australian synchrotron is presented here. The method demonstrates preparation of the sample, including the transfer of lysozyme crystals into a high viscous media (silicone grease), and the operation of the injector for data collection at MX2.

The goal of this protocol is to demonstrate how to prepare serial crystallography samples for data collection on a high viscous injector, Lipidico, recently commissioned at the Australian synchrotron.

Protocolo de inyección lipídica para mediciones de cristalografía en serie en el sincrotrón australiano

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built ...

Blood Flow Imaging with Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

Imágenes de flujo sanguíneo con Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...

Establishing an Octopus Ecosystem for Biomedical and Bioengineering Research

Many developments in biomedical research have been inspired by discovering anatomical and cellular mechanisms that support specific functions in different species. The octopus is one of these exceptional animals that has given scientists new insights into the fields of neuroscience, robotics, regenerative medicine, and prosthetics. Research with this species of cephalopods requires the set-up of complex facilities and intensive care for both the octopus and its ecosystem that is critical for the project&#39;s success. This system requires multiple mechanical and biological filtering systems to provide a safe and clean environment for the animal. Along with the control system, specialized routine maintenance and cleaning are required to effectively keep the facility operating long term. It is advised to provide an enriched environment to these intelligent animals by changing the tank&#39;s landscape, incorporating a variety of prey, and introducing challenging tasks for them to work through. Our results include MRI and a whole-body autofluorescence imaging as well as behavioral studies to better understand their nervous system. Octopuses possess unique physiology that can impact many areas of biomedical research. Providing them with a sustainable ecosystem is the first crucial step in uncovering their distinct capabilities.

Understanding the unique physiological and anatomical structures of octopuses can greatly impact biomedical research. This guide demonstrates how to set-up and maintain a marine environment to accommodate this species and includes state-of-the-art imaging and analytical approaches to visualize octopus' nervous system anatomy and function.

Establecimiento de un ecosistema de pulpos para la investigación biomédica y de bioingeniería

Many developments in biomedical research have been inspired by discovering anatomical and cellular mechanisms that support specific functions in different ...