Damos las gracias al Centro de Desarrollo Biológico de imágenes (BIDC) Universidad de California, San Francisco para el uso de la Imaris, XT Imaris y Matlab. Damos las gracias a V. Kharazia por la asistencia técnica y AT Henry, LK Floren, L. Daitch por sus contribuciones a la edición del manuscrito. Este trabajo fue apoyado por fondos del Estado de California de Investigación Médica sobre el Abuso de Alcohol y Sustancias a través de la UCSF para SEB, los Institutos Nacionales de Salud: 1R21DA029966-01 y el premio NIH vía rápida para examinar la colección MLSMR a SEB Escuela de Farmacia de la UCSF ( Trabajo de Directores y Farmacia Clínica) y la Escuela de Medicina (Farmacología Clínica y Terapéutica Experimental) para CLHK.

1. Three-dimensional Análisis morfométrico de una sola célula cambios fenotípicos <ol><li> Embrionarias humanas de riñón (HEK293) células fueron transfectadas con hemaglutinina (HA)-etiquetados factor liberador de corticotropina receptor-2 (CRF-R2), una proteína G-receptor acoplado (GPCR) como se ha descrito previamente 4, 5. </li><li> Las células se dejaron sin tratar (sin tratamiento, NT), estimuladas con el ligando endógeno CRF-R2, factor de liberación de corticotropina, CRF (1 mM, 30 min), o pretratadas con un selectivo antagonista de CRF-R2, anti-sauvagina 30 (como se -30, 1 mM, 30 min) antes del tratamiento con agonista. </li><li> Las células se fijaron, permeabilizaron y tratados con anti-HA. CRF-R2 fue visualizado utilizando Alexa 594 nm conjugado anti-ratón (IgG 1) anticuerpo. DAPI se utiliza para visualizar la fase mitótica núcleos. </li><li> Para limitar la subjetividad experimentador, las condiciones experimentales no se conocerá hasta después de que las imágenes fueron tomadas y analizadas. </li><li> Adquirimos la imagens de fijos HEK293 células por medio de un Plan apocromático 63x/1.4 aceite objetivo DIC y Zeiss LSM 510 microscopio confocal META conectado a un sistema integrado coherente láser de dos fotones compuesto por un láser de Verdi-V5 y un F-900 Mira sistema láser. </li><li> Durante el proceso de adquisición de datos, las células fueron compartimentadas tanto por seccionamiento multiespectral, 488 nm, y 790 nm (~ 350 nm Ex 2ph.) Y z-partición (0,5 incrementos mu m) para incluir los datos de la membrana nuclear hasta el receptor extracelular exterior extremidades. </li><li> Los datos de fluorescencia se procesaron primero usando Imaris, que permite la visualización y la segmentación de conjunto de datos de microscopía 3D, y un modelo 3D compuesto de voxels cúbicos se ha creado para el análisis morfométrico. Entonces, Imaris XT módulo se utilizó para Imaris de interfaz con el lenguaje MATLAB programa de ordenador para determinar el punto de coordenadas de las extensiones de GPCR. Para tener en cuenta la variabilidad celular, se obtuvieron y analizaron imágenes de fluorescencia taken 22 de las células: sin tratamiento (NT) (n = 7), agonista (CRF) tratamiento (n = 8) y pre-tratamiento con antagonista (AS-30) antes del tratamiento con agonista (n = 7) (Figura 2) . <ol><li> La región de interés (ROI) debe incluir una célula que no está en la etapa mitótica activo y no está cerca de otras células. De esta manera, el análisis se incluyen las células con un solo núcleo y las extensiones de los receptores no son perturbadas por la proximidad de otras células. </li><li> La estructura 3D celular fue reconstruida primero multiespectrales de los datos de fluorescencia utilizando Imaris (v.7.1.1). </li><li> Tras el algoritmo diseñado por Imaris, la primera representación de la superficie se utiliza para representar la membrana nuclear. Imaris determinará si hay más de un núcleo en la ROI. </li><li> Entonces, el algoritmo de creación de puntos se utiliza para localizar la extensión CRF-R2. Detección de puntos se utilizó ya que compensa el ruido de fondo y la intensidad irregular de lacompleja red de células en forma amorfa. </li><li> Para maximizar la inclusión de cada unidad de detección de fluorescencia de CRF-R2, diámetro de los puntos &quot;se fijó a 0,2 micras, que es la unidad más pequeña dentro de la imagen para extrapolar la información distinta en la forma de una intensidad medida usando un filtro de Gauss. Lugar filtrado se incorporó en el proceso de creación de spots automatizado. El software, sin embargo, proporciona al usuario la flexibilidad de utilizar filtros para definir los parámetros. </li><li> Para evitar el truncamiento de datos, el conjunto de datos se convierten de 8-bits (sin signo) de punto fijo, a decimal de 32-bit. </li><li> Los datos de intensidad del voxel fueron intercambiados para detectar datos de coordenadas utilizando Imaris XT módulo interfaz con MATLAB y la ubicación espacial exacta de cada punto se determinó mediante la realización de una transformación a distancia utilizando la membrana nuclear como punto de referencia (Figura 3). </li></ol></li><li> Los datos resultantes se pueden cuantificar y presentan en formato gráfico para sanálisis tatistical. Las comparaciones entre grupos se realizaron mediante ANOVA de dos vías y post-test de Bonferroni. Los datos se presentan como media ± desviación estándar. Las diferencias se consideraron significativas a * p &lt;0,05. Los cálculos se realizaron con el programa GraphPad Prism 5,02 (Figura 4). </li></ol> 2. Los resultados representativos Para demostrar la potencia de nuestro enfoque, se cuantificó los cambios celulares que resultan de la interacción de proteína G-junto receptores (GPCR) y el factor de liberación de corticotropina receptor-2 (CRF-R2) con su ligando endógeno CRF en células HEK293 transfectadas. Se demuestra que el CRF-R2 receptores están localizados en la membrana plasmática y se proyectan desde las regiones finitas de la membrana de las células (Figura 2A y Movie 1). Utilizando el análisis 2D convencional, es posible detectar este subconjunto de extracelulares CRF-R2 receptores sólo si se analiza la adhesión receptor points en el cristal cubre. Por lo tanto, perdemos cualquier otra información derivada de z apilados datos multiespectrales (Figura 5). Cuando las células se tratan con CRF, los receptores extracelulares se reducen considerablemente, como se muestra por la disminución de la distancia de los puntos de la membrana plasmática. También se redistribuye desde ubicaciones principalmente finitos en un número de localizaciones diferenciadas (Figura 2B y Película 2). El efecto del CRF en la distribución del receptor de membrana es impedido por el pretratamiento con el antagonista del CRF-R2 específico, antisavagine 30 (AS-30) y nos encontramos con que las extensiones CRF-R2 no cambia (Figura 2C y Movie 3). La distribución distal de manchas, trazados en las 5 micras de color espectro-intervalos codificados, se utiliza para visualizar la distancia de los vóxeles de la membrana nuclear. No pretreatm tratamiento y antagonistaent (AS-30, 1 mM, 30 min) antes del tratamiento con el agonista (CRF, 1 mM, 30 min) no muestran ninguna significativa (ns) diferencia en la contracción de GPCR. El tratamiento de las células con el agonista (CRF, 1 mM, 30 min) reduce progresivamente la cantidad de CRF-R2 que contienen voxels en comparación con ningún tratamiento, 0-5 micras (ns), 6-15 micras (** p &lt; 0,01) y&gt; 15 micras (*** p &lt;0,005), o en comparación con AS-30 tratamiento, 0-10 micras (ns), 11-15 micras (** p &lt;0,01) y&gt; 15 micras (*** p &lt;0,005) (Figura 4). <img alt="Figura 1" src="/files/ftp_upload/4233/4233fig1.jpg" /> Figura 1. Esquema de las técnicas actuales disponibles y su limitación para analizar imágenes de fluorescencia. <a href="http://www.jove.com/files/ftp_upload/4233/4233fig1large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> . <img alt="Figura 2" src="/files/ftp_upload/4233/4233fig2.jpg" /> <strong&gt; Figura 2. 3D imágenes multiespectrales de fluorescencia de las células HEK293 transfectadas con HA-CRF-R2 antes y después de agonista y antagonista de tratamiento. Imágenes fusionadas que muestran HA-CRF-R2-probaron utilizando anti-HA y se visualizaron utilizando Alexa 488 conjugada con anticuerpo anti-ratón (IgG 1) secundaria; DAPI se utiliza para visualizar los núcleos. Las imágenes fueron obtenidas con el láser confocal de barrido (CLS) microscopio. Barra de escala 5 micras. <img alt="Figura 3" src="/files/ftp_upload/4233/4233fig3.jpg" /> Figura 3. Modelo 3D de células HEK293 transfectadas con HA-CRF-R2 reconstruido a partir de las imágenes utilizando software CLS Imaris. Superficie de representación del núcleo y la creación de manchas describir extensiones GPCR convertidos en pequeñas vesículas. Los datos de fluorescencia se procesaron primero usando Imaris que permite la visualización y la segmentación de la microscopía 3D conjunto de datos. Luego, Imaris XT se utilizó para Imaris interfaz con MATLAB. La intensidad del voxel se intercambiaron enlugar coordina. Las manchas de color espectro-codificados (azul 0-5 micras, verdes, amarillas 6-10 m, 11-15 micras y rojo&gt; mu m 15) representan la forma de la distancia de la membrana nuclear. Escala de 5 micras. <img alt="Figura 4" src="/files/ftp_upload/4233/4233fig4.jpg" /> Figura 4. Representación gráfica de la distribución distal de puntos trazados en el espectro de color codificado en intervalos de 5 micras se utiliza para visualizar la distancia de los vóxeles de la membrana nuclear. Ningún tratamiento y pretratamiento con un antagonista (AS-30, 1 mM, 30 min) antes del tratamiento agonista (CRF, 1 mM, 30 min) no muestran ninguna significativa (ns) diferencia en la contracción GPCR. El tratamiento de las células con agonista (CRF, 1 mM, 30 min) reduce progresivamente la distancia del número de CRF-R2 que contienen voxels en comparación con ningún tratamiento 0-5 micras (ns), 6-15 micras (** p &lt;0,01) y&gt; 15 micras (*** p &lt;0,005), o como tratamiento-30 0-10 micras (ns), 11-15<html> m (** p &lt;0,01) y&gt; 15 micras (*** p &lt;0,005). <img alt="Figura 5" src="/files/ftp_upload/4233/4233fig5.jpg" /> Figura 5. Limitación del análisis 2D morfométrico de las células HEK 293 transfectadas con HA-CRF-R2. La sección del plano medio de las células (3-4 micras por encima del cubreobjetos de vidrio) que muestra el centro de los núcleos se visualizaron con DAPI y HA-CRF R2-probaron utilizando anti-HA y se visualizaron utilizando Alexa 488 nm conjugado anti-ratón (IgG 1) secundaria anticuerpo y adquirida con CLS no muestra ninguna diferencia entre (A) sin tratamiento (NT) y (B) agonista (CRF, 1 mM, 30 min), mientras que los puntos de adherencia del receptor son dramáticamente diferentes. Película 1. Giratorio modelo 3D en el &quot;superar&quot; el modo de células HEK 293 transfectadas con HA-CRF-R2, ningún tratamiento, para evaluar las diferencias fenotípicas celulares entre las proteínas receptoras. Bar escala de 5 a 20 micras./ Files/ftp_upload/4233/4233movie1.avi &quot;target =&quot; _blank &quot;&gt; Haga clic aquí para ver la película. Movie 2. Giratorio modelo 3D en el &quot;superar&quot; el modo de células HEK 293 transfectadas con HA-CRF-R2, el tratamiento con agonistas, CRF (CRF, 1 mM, 30 min) para evaluar las diferencias fenotípicas celulares entre las proteínas receptoras. Bar escala de 5 a 20 micras. <a href="http://www.jove.com/files/ftp_upload/4233/4233movie2.avi" target="_blank">Haga clic aquí para ver la película.</a> Película 3. Giratorio 3D en el &quot;superar&quot; modo de células HEK 293 transfectadas con HA-CRF-R2, el pretratamiento con un antagonista (AS-30, 1 mM, 30 min) antes del tratamiento agonista (CRF, 1 mM, 30 min ) para evaluar las diferencias entre los fenotipos celulares proteínas receptoras. Bar escala de 5 a 20 micras. <a href="http://www.jove.com/files/ftp_upload/4233/4233movie3.avi" target="_blank">Haga clic aquí para ver la película.</a>

<ol>
	<li>West, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design-based+stereological+methods+for+counting+neurons.">Design-based stereological methods for counting neurons.</a> Prog, Brain Res. 135, 43-51 (2002).</li><li>Burke, M., Zangenehpour, S., Mouton, P. R., Ptito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knowing+what+counts:+unbiased+stereology+in+the+non-human+primate+brain.">Knowing what counts: unbiased stereology in the non-human primate brain.</a> J. Vis. Exp. (27), e1262 (2009).</li><li>Sugawara, Y., Ando, R., Kamioka, H., Ishihara, Y., Honjo, T., Kawanabe, N., Kurosaka, H., Takano-Yamamoto, T., Yamashiro, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+three-dimensional+morphometry+and+cell-cell+communication+of+the+osteocyte+network+in+chick+and+mouse+embryonic+calvaria.">The three-dimensional morphometry and cell-cell communication of the osteocyte network in chick and mouse embryonic calvaria.</a> Calcif. Tissue Int. 88, 416-424 (2011).</li><li>Vickery, R. G., von Zastrow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+dynamin-dependent+and+-independent+mechanisms+target+structurally+homologous+dopamine+receptors+to+different+endocytic+membranes.">Distinct dynamin-dependent and -independent mechanisms target structurally homologous dopamine receptors to different endocytic membranes.</a> J. Cell Biol. 144, 31-43 (1999).</li><li>Bartlett, S. E., Enquist, J., Hopf, F. W., Lee, J. H., Gladher, F., Kharazia, V., Waldhoer, M., Mailliard, W. S., Armstrong, R., Bonci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+responsiveness+is+regulated+by+targeted+sorting+of+D2+receptors.">Dopamine responsiveness is regulated by targeted sorting of D2 receptors.</a> Pro.c Natl. Acad. Sci. U.S.A. 102, 11521-11526 (2005).</li><li>Gordon, A., Colman-Lerner, A., Chin, T. E., Benjamin, K. R., Yu, R. C., Brent, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+quantification+of+molecules+and+rates+using+open-source+microscope-based+cytometry.">Single-cell quantification of molecules and rates using open-source microscope-based cytometry.</a> Nat. Methods. 4, 175-181 (2007).</li><li>Schock, F., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+epithelial+morphogenesis.">Molecular mechanisms of epithelial morphogenesis.</a> Annu. Rev. Cell Dev. Biol. 18, 463-493 (2002).</li><li>Pincus, Z., Theriot, 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=Comparison+of+quantitative+methods+for+cell-shape+analysis.">Comparison of quantitative methods for cell-shape analysis.</a> J. Microsc. 227, 140-156 (2007).</li><li>Spiller, D. G., Wood, C. D., Rand, D. A., White, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+single-cell+dynamics.">Measurement of single-cell dynamics.</a> Nature. 465, 736-745 (2010).</li><li>Loo, L. H., Wu, L. F., Altschuler, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-based+multivariate+profiling+of+drug+responses+from+single+cells.">Image-based multivariate profiling of drug responses from single cells.</a> Nat Methods. 4, 445-453 (2007).</li><li>Yarrow, J. C., Totsukawa, G., Charras, G. T., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+cell+migration+inhibitors+via+automated+microscopy+reveals+a+Rho-kinase+inhibitor.">Screening for cell migration inhibitors via automated microscopy reveals a Rho-kinase inhibitor.</a> Chem. Biol. 12, 385-395 (2005).</li></ol>

No hay conflictos de interés declarado.

Hemos demostrado que el tratamiento CRF induce un cambio significativo en la morfología y ubicación de CRF-R2. El cambio en el CRF-R2 fue inhibido por el tratamiento antagonista selectivo. Hemos demostrado que las modificaciones no se detectaron receptores y no puede ser medido utilizando el estándar de las técnicas multiespectrales 2D. La capacidad para estudiar complejos imágenes en 3D es crítica para incorporar la complejidad de los parámetros biológicos para el análisis morfométrico. Hemos sido capaces de ...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> De riñón embrionario humano (HEK293) </td><td> American Type Culture Collection </td><td> CRL-1573 </td><td></td></tr><tr><td> Dulbecco Eagle modificado (DMEM) </td><td> Invitrogen </td><td> 11965118 </td><td></td></tr><tr><td> Suero bovino fetal (FBS) </td><td> Invitrogen </td><td> SH30070.03 </td><td></td></tr><tr><td> AlexaFluor-488 (IgG2b) </td><td> Invitrogen </td><td> A-11001 </td><td></td></tr><tr><td> monoclonal anti-HA.11 (IgG 1) </td><td> Covance </td><td> 16B12 </td><td></td></tr><tr><td> DAPI </td><td> Vector Laboratories </td><td> H-1200 </td><td>; </td></tr><tr><td> CRF </td><td> Sigma </td><td> C2917 </td><td></td></tr><tr><td> Antisauvagine-30 (TAL-30) </td><td> Sigma </td><td> A4727 </td><td></td></tr></tbody></table>

an analytical tool that quantifies cellular morphology changes from three-dimensional fluorescence images

Las herramientas de software más comunes de análisis disponibles para la medición de imágenes de fluorescencia son para datos de dos dimensiones (2D) que dependen de los ajustes manuales para la inclusión y la exclusión de los puntos de datos, y asistido por ordenador de reconocimiento de patrones para facilitar la interpretación y el análisis de resultados. Se ha convertido cada vez más importante para ser capaz de medir las imágenes de fluorescencia construidas a partir de conjuntos de datos tridimensionales (3D) con el fin de ser capaz de capturar la complejidad de la dinámica celular y entender la base de la plasticidad celular dentro de los sistemas biológicos. Instrumentos sofisticados microscopía han permitido la visualización de 3D imágenes de fluorescencia a través de la adquisición de imágenes multiespectrales de fluorescencia y un software de análisis de gran alcance que reconstruye las imágenes de confocal pilas que luego proporcionan una representación 3D de las imágenes recogidas en 2D. Diseño avanzado de los métodos basados ​​en estereología han pasado de la aproximación y los supuestos de la originaciónl modelo basado estereología 1 incluso en secciones de tejidos complejos 2. A pesar de estos avances científicos en microscopía, permanece una necesidad de un método analítico automatizado que explota completamente los datos 3D intrínsecas para permitir el análisis y la cuantificación de los complejos cambios en la morfología celular, la localización de proteínas y tráfico de receptores. Las técnicas actuales disponibles para cuantificar las imágenes de fluorescencia incluyen Meta-Morph (Molecular Devices, Sunnyvale, CA) y la imagen J (NIH), que proporcionan un análisis manual. Imaris (Andor Technology, Belfast, Irlanda del Norte) software proporciona la función de MeasurementPro, que permite la creación manual de los puntos de medición que se pueden colocar en una imagen de volumen o dibujado en una serie de cortes 2D para crear un objeto 3D. Este método es útil para mediciones de punto de un solo clic para medir una distancia en línea entre dos objetos o para crear un polígono que encierra una región de interés, pero es difícil de aplicar a complex estructuras de redes celulares. Filamentos trazador (Andor) permite la detección automática de la 3D neuronal filamento-como sin embargo, este módulo ha sido desarrollado para medir estructuras definidas, tales como neuronas, que se componen de dendritas, axones y espinas (estructura de árbol). Este módulo ha sido ingeniosamente utilizado para realizar mediciones morfológicas a las células no neuronales 3, sin embargo, los datos de salida de proporcionar información de una red celular extendida mediante el uso de un software que depende de una forma de la célula definido en lugar de ser un modelo celular amorfo en forma. Para superar el problema de analizar amorfo en forma de células y hacer el software más adecuado para una aplicación biológica, Imaris desarrollado célula Imaris. Este fue un proyecto científico con la Eidgenössische Technische Hochschule, que ha sido desarrollado para calcular la relación entre las células y orgánulos. Mientras que el software permite la detección de las limitaciones biológicas, forzando un núcleo por célulay el uso de las membranas celulares de las células del segmento, no se puede utilizar para analizar los datos de fluorescencia que no son continuas, porque se construye idealmente superficie celular sin espacios vacíos. Para nuestro conocimiento, en la actualidad no modificable por el usuario enfoque automatizado que proporciona información morfométrica de las imágenes de fluorescencia 3D ha sido desarrollado que logra información celular espacial de una forma indefinida (Figura 1). Hemos desarrollado una plataforma analítica usando el módulo principal de software Imaris y XT Imaris interfaz con MATLAB (Mat Works, Inc.). Estas herramientas permiten la medición 3D de las células sin una forma predefinida y con inconsistentes componentes de la red de fluorescencia. Además, este método permitirá a los investigadores que han extendido experiencia en sistemas biológicos, pero no familiaridad con las aplicaciones informáticas, para llevar a cabo la cuantificación de los cambios morfológicos en la dinámica celular.

Watch this Scientific Journal Video about An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images at JoVE.com

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

Hemos desarrollado una plataforma de software que utiliza Imaris Neuroscience, ImarisXT y MATLAB para medir los cambios en la morfología de una forma indefinida tomado de tres dimensiones de fluorescencia confocal de células individuales. Este nuevo enfoque se puede utilizar para cuantificar los cambios en la forma celular después de la activación del receptor y por lo tanto representa una posible herramienta adicional para el descubrimiento de fármacos.

Una herramienta de análisis que cuantifica los cambios celulares Morfología de imágenes tridimensionales de fluorescencia

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

an-analytical-tool-that-quantifies-cellular-morphology-changes-from

Research

JoVE Journal

Biology

14.5K vistas.  University of California, San Francisco. Hemos desarrollado una plataforma de software que utiliza Imaris Neuroscience, ImarisXT y MATLAB para medir los cambios en la morfología de una forma indefinida tomado de tres dimensiones de fluorescencia confocal de células individuales. Este nuevo enfoque se puede utilizar para cuantificar los cambios en la forma celular después de la activación del receptor y por lo tanto representa una posible herramienta adicional para el descubrimiento de fármacos.

Video: An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

Basada en el chip tridimensional de cultivo celular en perfundido Micro-biorreactores

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

Investigación

Biología

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Microfabricación de chip del tamaño de andamios para el cultivo de células en tres dimensiones

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

Un método de alto rendimiento a nivel mundial para estudiar la morfología orgánulo en S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

Concentración, la carga, y la alineación de una célula de ultracentrifugación analítica de muestras

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.

Los cambios en la morfología de la glándula mamaria y el riesgo de cáncer de mama en ratas

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Aislamiento de células epiteliales mamarias de tridimensional mixto de células Esferoide Co-cultura

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

Análisis rápido y exploración de microscopía de fluorescencia de Imágenes

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET Imaging en hidrogeles tridimensionales

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-claro: Un tejido claro método de proyección de imagen tridimensional del tejido adiposo

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

Enfoques cuantitativos para el estudio de las estructuras celulares y la morfología de los organélilos en Los cenoheman

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...