Reconocemos los donativos en especie de las cepas bacterianas de los grupos de la Prof. Judith Armitage (Universidad de Oxford, Reino Unido) y el Prof. Conrad Mullineaux (Universidad Queen Mary de Londres, Reino Unido). IMD es financiado conjuntamente por el Departamento de Bioquímica (Universidad de Oxford) y OCISB; AR es financiado por una Ingeniería y Ciencias Físicas Research Council (EPSRC) becas para estudios de DTC, ND se financia con la Biotecnología y Ciencias Biológicas de Investigación (BBSRC), MCL es financiado por una beca Real Sociedad de Investigación de la Universidad.

<ol><li> Para iniciar este procedimiento, 50 l de material congelado de la proteína fluorescente expresando Escherichia coli células bacterianas primero se descongela y se cultiva aeróbicamente con agitación en 5 ml de medio de cultivo LB noche a 37 º C. Por la mañana, 50 l de esta cultura saturada se extrae y sub-cultivadas en un mínimo de M63 medios de glucosa en la cultura, la incubación a 30 º C durante 4 a 6 horas. Aquí se demuestra el uso de dos cepas diferentes de células, una de las cuales expresa un transporte de electrones del citocromo fusionado a GFP, y el otro que expresa una proteína implicada en el motor flagelar bacteriano fusionado a GFP. </li><li> Células o bien puede recogidas directamente del crecimiento sub-cultura, si los ve como muestras inmovilizadas, o pueden ser cortados para truncar los flagelos bacterianos ver si en &quot;atar&quot; las condiciones. </li><li> Esquila consiste en colocar por lo general 1-5 ml de la sub-cultura en un dispositivo que consta de dos jeringas estériles se unió por un tubo estéril. El corte se realiza por la alternancia de empujar en cada bomba de jeringa para impulsar la cultura a través del tubo estrecho. Esto se hace 50-100 veces, dependiendo de la extensión del corte requerido. La cultura se centrifuga para sedimentar las células, que se vuelve a suspender en medios mínimos para eliminar los fragmentos de los flagelos. </li><li> A continuación, preparar limpiar BK7 cubreobjetos de vidrio por inmersión en una solución saturada de KOH en etanol durante 20 minutos, luego enjuagar bien en agua desionizada y etanol y dejar secar al aire durante al menos 1 h. </li><li> Construimos una simple célula de flujo a la casa de las células en el microscopio. Esto implica el trazado de líneas de grasa de parafina en un portaobjetos de vidrio BK7 microscopio y luego la creación de un túnel-sandwich, colocando uno de los coverlips limpia en la parte superior, y presionando suavemente con un par de pinzas, dando un volumen de celda de flujo de 5.10 l . </li><li> Para observar las células inmovilizadas llenamos el flujo de las células mediante una inyección con un 0,1% w / v solución de poli-L-lisina, y deje que se incuban a temperatura ambiente durante al menos 1 min. Luego enjuagar con 100 medios mínimos l mediante la inyección de los medios de comunicación de un extremo de la celda de flujo y transporte de humedad con papel de seda de la otra. </li><li> A continuación, la mecha a través de 20 l de una dilución 1:500 de 200 nm de diámetro microesferas de látex (Polysciences) en medios mínimos para marcar la superficie cubreobjetos. La celda de flujo se invierte de tal manera que el cubreobjetos se encuentra hacia abajo, colocar en una plataforma clara de la superficie en una cámara sencilla de humedad, y se incuban a temperatura ambiente durante 5 min. Cuentas no consolidadas se eliminan por lavado por absorción a través de 100 medios mínimos l. </li><li> Si queremos observar células atados omitimos el paso de poli-L-lisina y en lugar de llenar la celda de flujo con 5 mg / ml de anticuerpos anti-flagelina. La celda de flujo es un lugar en la cámara de humedad durante 10 minutos y luego se eliminan a través de absorción. </li><li> 20 l de cultivo de células es entonces malo a través de la celda de flujo, ya sea usando la muestra cortada si la observación de las células atados o la muestra unsheared en ver células inmovilizadas. </li><li> La celda de flujo se invierte y se coloca en la cámara de humedad durante 20 minutos. Las células no unidas se lavan a cabo por absorción a través de 100 medios mínimos l. </li><li> Una gota de aceite de inmersión se coloca en el centro de la superficie superior del cubreobjetos y el flujo de las células se coloca suavemente sobre el soporte de muestras de la costumbre microscopio de fluorescencia construido, haciendo contacto con la óptica de lentes de alta apertura numérica objetivo. </li><li> El microscopio electrónico, multiplicando la cámara se enciende y la cámara en que se enfrió a -70 º C, el software está listo para adquirir imágenes a una velocidad típica de 25 Hz en modo de cuadro de transferencia, en un principio disenabling el control de ganancia en el cámara. </li><li> La iluminación de campo claro está encendido y la imagen se pusieron sobre la mesa, la selección de una célula adecuada o de un grupo de células para formar una imagen sobre la base de su ser atrapado con su eje paralelo a la superficie a largo cubreobjetos. El objetivo es ajustar con precisión para asegurar que las 200 cuentas nm látex pegado a la superficie del cubreobjetos son sólo en el enfoque. </li><li> Una secuencia de imágenes se adquieren en el campo claro para grabar el contorno del cuerpo celular. La iluminación de campo claro se apaga y la ganancia de la cámara se activa al máximo. </li><li> Para una adquisición estándar mediante fluorescencia de reflexión total interna (TIRF o) un rayo láser en las longitudes de onda (en este caso, para la excitación de la proteína verde fluorescente, se utiliza un láser nm 473) se pre-establecido con el fin de centrarse en el plano focal posterior de la lente del objetivo, pero desplazados lateralmente desde el eje óptico para generar un campo evanescente de excitación de fluorescencia en la muestra de células. </li><li> La adquisición de la cámara se inicia y se abre el obturador de láser para excitar a las proteínas fluorescentes en las bacterias. Los parámetros de la intensidad del láser y la velocidad de adquisición deben ser optimizados para el sistema biológico particular bajo investigación por experimentar con diferentes valores, pero un típico rango rElevant al estudio de los complejos móviles proteína en la membrana celular son la energía láser 1.10 mW en una zona de excitación circular de diámetro aproximadamente 30 micras, con un tiempo de exposición por cuadro de 50 a 40 ms. Las muestras se iluminan hasta photobleached, por lo general de unos 10 s. </li><li> Este protocolo se puede utilizar tanto para las células atados en la que el cuerpo de la célula está girando alrededor de un punto de unión entre el cubreobjetos y un talón de flagelos, y por células inmovilizadas que están rígidamente fijado a la superficie cubreobjetos. </li><li> Algunas cepas de células que tienen un alto número de copias de los complejos se benefician de un láser blanqueador inicial limitada por difracción se centraron en un polo de la célula antes de la TIRF imágenes. Este blanqueo es equivalente a la utilizada en la recuperación de la fluorescencia después de photobleaching (o FRAP). Utilizando nuestro microscopio costumbre parte de la luz láser de excitación puede ser alimentado de en una segunda vía independiente utilizada para el blanqueo de tipo FRAP. Por lo general 1-10 mW de potencia láser se utiliza, con un tiempo de blanqueo típica en el rango de 10 a 300 ms. Esto se traduce en contraste de imagen mucho mayor en la zona de blanqueado de la célula que permite complejos individuales que posteriormente se difunden en la zona a ser más fácil de visualizar. </li><li> Para visualizar los complejos en el citoplasma de la célula que se difunden mucho más rápido que los de la membrana celular se emplea un modo de iluminación diferente llamado &quot;slimfield&quot;. Aquí un láser enfocado es expandido lateralmente para abarcar una sola célula. Esto produce un campo muy intenso que permite la exposición mucho más rápido de lo general una milésima de segundo que deban tomarse. </li><li> Tras la adquisición de datos, las imágenes se introducen en el software personalizado por escrito (codificado en LabVIEW 8.5). Este detecta automáticamente las posiciones de puntos fluorescentes en las células con una precisión de unos pocos nanómetros general y los extractos de su tamaño y brillo. El brightnessof es el trazo photobleaching con respecto al tiempo de un complejo molecular seguimiento para estimar la estequiometría, es decir, la cantidad de proteínas fluorescentes individuales constituyen un complejo molecular única. </li></ol> Los resultados representativos: Cuando el protocolo se hace correctamente las imágenes de las células vistas en campo claro es muy distinto, con los perímetros de los cuerpos celulares oscuro contra un cuerpo de la célula blanco / gris (Figura 1a). En la fluorescencia utilizando células inmovilizadas, podemos ver la intensidad de los puntos distintos, por lo general de 250 a 300 nm de ancho (Figura 1b). Las células sanas, sujetos, se verá a girar alrededor del punto de unión de sujeción en las imágenes de campo claro. En virtud de excitación de fluorescencia algunos complejos moleculares, en nuestro caso también podría ser visto en el punto de unión, lo que indica una localización de la proteína marcada con el motor flagelar. Estos puntos son complejos moleculares individuales y el número de visto dependerá del modo de iluminación utilizados y cómo muchos de los complejos están presentes en la célula en un momento dado. La movilidad de los lugares depende del sistema biológico específico en estudio. Si la densidad de puntos es inicialmente muy alto, como es el caso de los citocromos etiqueta se utiliza aquí, a continuación, realizar un blanqueador FRAP inicial puede mejorar el contraste de la imagen. <img src="/files/ftp_upload/1508/1508Figure1tmb.jpg" border="0" alt="Figura 1" /> Figura 1. (A) claro y (B) la imagen TIRF (falso color) de una célula de Escherichia coli inmovilizada expresar una proteína de fusión a la proteína verde fluorescente (GFP), que se sabe están involucrados en los motores de flagelos de las bacterias. Por favor, <a href="https://www.jove.com/files/ftp_upload/1508/1508Figure1.jpg">haga clic aquí</a> para ver una versión ampliada de la figura 1.

<ol>
	<li>Leake, M. C., Chandler, J. H., Wadhams, G. H., Bai, F., Berry, R. M., Armitage, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometry+and+turnover+in+single,+functioning+membrane+protein+complexes.">Stoichiometry and turnover in single, functioning membrane protein complexes.</a> Nature. 443, 355-358 (2006).</li><li>Leake, M. C., Greene, N. P., Godun, R. M., Granjon, T., Buchanan, G., Chen, S., Berry, R. M., Palmer, T., Berks, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable+stoichiometry+of+the+TatA+component+of+the+twin-arginine+protein+transport+system+observed+by+in+vivo+single-molecule+imaging.">Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging.</a> Proc Natl Acad Sci U S A. 105, 15376-15381 (2008).</li><li>Leake, M. C., Wilson, D., Gautel, M., Simmons, R. M., 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=The+elasticity+of+single+titin+molecules+using+a+two-bead+optical+tweezers+assay.">The elasticity of single titin molecules using a two-bead optical tweezers assay.</a> Biophys. J. 87, 1112-1135 (2004).</li><li>Leake, M. C., Wilson, D., Bullard, B., Simmons, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elasticity+of+single+kettin+molecules+using+a+two-bead+laser-tweezers+assay.">The elasticity of single kettin molecules using a two-bead laser-tweezers assay.</a> FEBS Lett. , 535-555 (2003).</li><li>Lenn, T., Leake, M. C., Mullineaux, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+clustering+and+dynamics+of+cytochrome+bd+complexes+in+the+Escherichia+coli+plasma+membrane.">In vivo clustering and dynamics of cytochrome bd complexes in the Escherichia coli plasma membrane.</a> Mol. Microbiol. 70, 1397-1407 (2008).</li><li>Lenn, T., Leake, M. C., Mullineaux, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+Escherichia+coli+OXPHOS+complexes+concentrated+in+specialised+zones+within+the+plasma+membrane.">Are Escherichia coli OXPHOS complexes concentrated in specialised zones within the plasma membrane.</a> Biochem. Soc. Trans. 36, 1032-1036 (2008).</li><li>Lo, C. J., Leake, M. C., Pilizota, T., Berry, R. M. <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+measurements+of+Membrane+Potential,+Sodium-Motive+Force+and+Flagellar+Motor+Speed+in+Escherichia+coli.">Single-cell measurements of Membrane Potential, Sodium-Motive Force and Flagellar Motor Speed in Escherichia coli.</a> Biophys. 93, 294-302 (2007).</li><li>Lo, C. J., Leake, M. C., Berry, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+measurement+of+intracellular+sodium+concentration+in+single+Escherichia+coli+cells.">Fluorescence measurement of intracellular sodium concentration in single Escherichia coli cells.</a> Biophys. J. 90, 357-3565 (2006).</li></ol>

The authors have nothing to disclose.

Se debe tener cuidado de no &quot;sobre corte&quot; de células para observar las bacterias atados, ya que esto puede perjudicar la funcionalidad de los motores del flagelo. Es importante la utilización de las células durante mucho más tiempo de una hora una vez en el portaobjetos del microscopio, ya que puede convertirse en falta de oxígeno. Optimización considerable puede ser requerido para encontrar las mejores condiciones de microscopio de imagen atiende a su sistema biológico específico bajo investigación. ...

visualizing single molecular complexes in vivo using advanced fluorescence microscopy

Una visión completa de los mecanismos de las células vivas sólo puede lograrse mediante la investigación de los procesos clave que provocan y dirigir los eventos a nivel celular. Hasta la fecha, la complejidad de corte de los sistemas biológicos ha provocado precisa una sola molécula de la experimentación a ser demasiado exigente, en lugar de centrarse en los estudios de sistemas de un solo uso a granel relativamente crudo media del conjunto de mediciones. Sin embargo, muchos procesos importantes ocurren en la célula viva en el ámbito de una o unas pocas moléculas, las mediciones del conjunto en general, la máscara de la naturaleza estocástica y heterogéneo de estos eventos. En este caso, mediante microscopía óptica avanzada y herramientas de análisis de análisis de imagen que se muestra cómo controlar las proteínas dentro de una sola célula viva bacteriana con una precisión de las moléculas individuales y cómo podemos observar la dinámica dentro de los complejos moleculares en el funcionamiento de máquinas biológicas. Las técnicas son directamente relevantes fisiológicamente. Son mínimamente perturbativa y no invasiva a la muestra biológica objeto de estudio y están totalmente adaptados para las investigaciones en la materia viva, las características no disponibles para los otros enfoques de una sola molécula de la biofísica. Además, las muestras biológicas estudiadas todos los productos de proteína fluorescente con etiquetas en niveles que son casi idénticas a las cepas de células no modificadas (&quot;codificación genómica&quot;), en comparación con el enfoque más común pero menos ideal para generar mucha más proteína que se producen de forma natural (&#39;plásmido de expresión &quot;). Por lo tanto, las muestras biológicas reales que serán investigados son significativamente más cerca de los organismos naturales, y por lo tanto, las observaciones más relevantes para los procesos fisiológicos reales.

Watch this Scientific Journal Video about Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy at JoVE.com

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Aquí nos demuestran los protocolos para la realización de una sola molécula de microscopía de fluorescencia en células vivas de las bacterias para permitir funcionales complejos moleculares para detectar, seguir y cuantificar.

Visualizar solo los complejos moleculares En Vivo Uso de microscopía de fluorescencia avanzada

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

visualizing-single-molecular-complexes-vivo-using-advanced

Research

JoVE Journal

Biology

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

9.2K vistas.  University of Oxford. Aquí nos demuestran los protocolos para la realización de una sola molécula de microscopía de fluorescencia en células vivas de las bacterias para permitir funcionales complejos moleculares para detectar, seguir y cuantificar.

Video: Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Visualización de una sola molécula de ADN de replicación con microscopía de fluorescencia

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Investigación

Biología

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Time-lapse de imágenes de mitosis después de la transfección de siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

Visualización de las bacterias en los nematodos mediante microscopía de fluorescencia

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Análisis cuantitativo de la autofagia mediante microscopía de fluorescencia avanzada 3D

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a &#34;double decker&#34; microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm.

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

The giant ciliate Stentor coeruleus is a classical system for studying regeneration and wound healing in single cells.&#160;By imaging Stentor cells simultaneously at low and high magnification it is possible to measure cytoplasmic flows before, during, and after wounding.

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

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 ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

La visualización de frecuencia Stromule con microscopía de fluorescencia

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

Proyección de imagen funcional de fábricas de transcripción Viral mediante microscopia de fluorescencia 3D

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

Visualización de la estructura de los ganglios linfáticos y la localización celular mediante la microscopía confocal Ex-Vivo

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Examen de la dinámica nuclear de levadura de ficción mitotica y meiótica por microscopía de células vivas de fluorescencia

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...