Este proyecto fue financiado por el CIHR y NSERC. Nos gustaría agradecer Jusiak Barb para contribuir a la creación de las cepas de moscas que expresan dsRed etiquetados SSR (línea BJ), y el Fondo para el UBC Bio-imágenes.

Parte 1: Preparación del tejido <ol><li> Nuestro objetivo es una preparación de tejidos de larvas de mosca en el sistema nervioso está intacto, pero la superficie interior de la pared muscular del cuerpo se expone a una hemolinfa artificial, y se pueden colocar cerca de un cubreobjetos microscopio para ópticas. En otras palabras, un interior a cabo la preparación de larvas. Dado que las preparaciones convencionales de tejidos se refieren al corte, fijación y estiramiento de la pared muscular del cuerpo, ya veces la eliminación de parte del sistema nervioso, necesitamos un enfoque diferente. Queríamos que los animales de dentro a fuera porque quería conseguir un buen vistazo a las características estructurales y los cambios estructurales en el tiempo en la sinapsis, y hemos querido mantener el sistema nervioso intacto. También hemos querido evitar el estiramiento del tejido, y la activación de los receptores de estiramiento, lo que haría que los músculos de contracción y la ruina de nuestras imágenes. </li> <li> Para empezar, el estadio de los animales. La alimentación de las larvas de tercer y errante larvas de tercera son grandes y fáciles de diseccionar lo que vamos a demostrar en W3 larvas. Este animal se tiene el fenotipo Tubby, y es muy amplia, por lo que es fácil de Evert. Hemos utilizado las larvas que vimos activamente gatear, incluyendo larvas de W3. </li><li> Limpie la superficie de un gusano con un pincel muy suave en una placa de Petri de H2O bidestilada. Las larvas tienen una mejor óptica limpia y la limpieza reduce las bacterias. </li><li> La transferencia de los animales a una pequeña placa de Petri de alrededor de 3 ml de hielo frío HL-6, y en la hemolinfa artificial. Coloque el plato en el hielo hasta que el animal deja de moverse, y se relaja (unos 5 minutos). </li><li> Sujete con fórceps punta muy fina en una mano y las tijeras de primavera en el otro. Yo uso las tijeras con la mano dominante. Haz un pequeño agujero en la pared del cuerpo con las tijeras para equlibrate la presión a través de la pared del cuerpo. </li><li> Sujetar al animal suavemente con las pinzas hacia abajo en la parte inferior de la placa y cortar la parte posterior de dos segmentos. Diseccionar las vísceras y la grasa corporal es probable que se mueven fuera de la cavidad del cuerpo. Cortar este tejido también. </li><li> Mantenga las larvas contra la parte inferior plato con unas pinzas finas. Mantenga un pin # 0 de insectos en (con punta roma) y empujarla contra la partes de la boca de las larvas. Empuje el aparato bucal a través de la cavidad del cuerpo como el que está girando un calcetín del revés. </li><li> El revés del tejido se parecerá a la figura: a continuación. Con ultra-fino de herramientas de disección, diseccionar los de grasa corporal y trachioles de la pared del cuerpo. Realmente difícil tratar de no tirar la trachioles, o desconectar el sistema nervioso. Rasga el trachioles rasgar los agujeros en la pared muscular del cuerpo. Extraer el cuerpo tanta grasa como sea posible. Ambas estructuras alterar la calidad óptica de sus tejidos. Es posible que desee omitir el café antes de hacer esta preparación. </li> <li> Cuando haya terminado el músculo será transparente, no opaco o blanco. Si ponemos la preparación de tejidos en HL-6 sin glutamato, a temperatura ambiente, es probable que se contraen rítmicamente, porque los generadores de patrón motor en el sistema nervioso central están trabajando. Evite el uso de preparaciones con los músculos contraídos, obviamente, o contratados irregularmente pared del cuerpo. El interior de la pared del cuerpo intacto fácilmente tiende a doblar por la mitad a lo largo de la línea media dorsal y ventral, por lo que la preparación le da a la izquierda oa la derecha &quot;hemi-animal&quot; para visualizar. </li><li> 1.9 Montar el tejido, ya sea en una cámara de pequeño volumen comercial, tales como la cámara de Warner, o un portaobjetos de microscopio con una disposición de cubierta antideslizante puente. Ver la parte 3 de sugerencias sobre el tejido de montaje. </li></ol> Parte 2: Etiquetado de Bouton neuronal con anticuerpos marcados con fluorescencia primaria contra el HRP. <ol><li> Poner 50 microlitros de la etiqueta fluorescente primario anti-cuerpo contra el HRP en HL-6 en una gota en una placa de Petri. Sumergir la preparación del cuerpo de adentro hacia afuera de la pared en el baño de tinte. Usted puede ver el etiquetado neurona terminal después de unos 5 minutos, pero se incuba durante 10-20 minutos, de una etiqueta completa y brillante. </li><li> Enjuague el tinte fuera durante 10-30 segundos en HL-6 a temperatura ambiente. No obligado tinte enjuaga rápidamente, así que el ciclo de enjuague no es necesario ser agresivo. </li><li> Variar la concentración de colorante y el tiempo de incubación, según sea necesario. </li></ol> Parte 3: etiquetado espacio Perisynaptic con dextrano conjugado flurophore <ol><li> Diluir con fluorescencia conjugada con dextrano tintura diluida en HL-6. Una concentración que funcionó bien para nuestros propósitos fue: </li><li> Si usted no está preocupado por el tiempo de acceso medio de contraste dentro de su espacio extracelular, poner una gota 20 micro litros de colorante en un &quot;puente&quot; cubreobjetos (véase la parte 4) y el depósito de preparación en el baño de tinte. </li></ol> Parte 4: Montaje del tejido para la visualización (con microscopía confocal). Si no es necesario para perfundir su preparación (breves observaciones) o si desea mantener el volumen de baño HL-6 pequeños, utilice el método de doble cara en puente Si desea perfundir su preparación, pruebe a usar una cámara de perfusión. Se utilizó un moCámara dified de instrumentos de Warner. Detalles para los dos siguientes. Montaje de preparación en un portaobjetos de doble puente. <ol><li> Use un portaobjetos muy limpio. Superglue dos cuadrados de 18 mm (# 1.5) cubreobjetos sobre el portaobjetos. Deje un espacio de 2 mm entre los bordes del cubreobjetos. </li><li> Deje que el pegamento seque completamente, o se forma una película extraña en los medios acuosos alrededor de su preparación. </li><li> Posición de la parte interior de larvas entre los bordes de los cubreobjetos. Es posible que necesite la posición de la preparación en una diagonal, si las larvas es grande y su sistema de adquisición de imágenes tiene una matriz de píxeles limitados. </li><li> Cubrir el tejido con un cubreobjetos # 1.5 mm, 18 mm (muy limpio). Se adhieren al &quot;top&quot; de la diapositiva colocada cubreobjetos se desliza el tejido de acompañamiento, con vaselina mínimo. </li><li> Ponga una gota de aceite de Cargill objetivo personalizado con un índice de refracción de 1,3379 (si se utiliza HL-6) en el cubreobjetos encima y voltear esta asamblea otra vez. </li><li> Coloque el conjunto del tejido en el microscopio de lado el petróleo, hacia el objetivo, y enfocará su objetivo. Montaje del tejido en una cámara de perfusión modificado. </li><li> Pegue un cubreobjetos de 1,5 a formar un piso en la cámara de RC 20. Coloque el tejido en la cámara de acuerdo con las instrucciones de RC-20. Use un pedazo de malla en lugar de Nytex un cubreobjetos para formar un techo de la cámara. </li></ol> Parte 5: Resultados del representante: <ol><li> De adentro hacia fuera la preparación del tejido disección de la secuencia: <img src="/files/ftp_upload/1154/fig1.jpg" alt="Figura 1" /> W3 larvas, se lavó (&quot;Tubby&quot; fenotipo) (5.1.1). W3 larvas, posterior dos segmentos disecados. Usted puede ver el cuerpo de grasa empujado fuera de la cavidad del cuerpo por cuerpo trans-presión de la pared (flecha). Trate de minimizar visceral &quot;erupción&quot;, haciendo un pequeño agujero en la pared del cuerpo un minuto antes de la disección (5.1.2). La preparación de las vísceras disecados antes de encender la preparación de adentro hacia afuera (5.1.3). La preparación, en su mayoría evertido, con el pasador (flecha) dentro de la luz de la preparación (5.1.4). El músculo se encuentra ahora en el exterior y la cutícula está en el interior, con alguna grasa corporal y trachioles adjunta (5.1.5). Un tejido completamente disecados preparación con casi todos los de la grasa corporal y trachioles disecados (5.1.6). <img src="/files/ftp_upload/1154/fig2a.jpg" alt="la figura 2" width="600" height="200" /></li><li> Vivir el etiquetado el uso de anti-HRP en la UNM larval. Un representante W3 larval músculos y nervios hacen sinapsis con una extensión gliales (de adentro hacia afuera-prep). Los terminales de las neuronas motoras bouton se etiquetan con un primario anti-cuerpo contra HRP (magenta), que se conjuga con Cy5. Los procesos gliales están etiquetados con las buenas prácticas agrarias (verde). <img src="/files/ftp_upload/1154/fig2b.jpg" alt="la figura 3" /></li><li> Espacio Perisynaptic fluorescencia visualiza con Alexa 680 dextrano en un revés de preparación. El proceso gliales (verde) está marcada con GFP membrana específicos. La República Socialista Soviética de post-sináptica en la superficie del músculo (azul) está marcado con dsRed. El dextrano Alexa (rojo) formas concentra en las zonas extracelular. El tinte de dextrano forma piscinas en forma de anillo en los espacios perisynaptic. El etiquetado de dextrano y SSR dsRed etiqueta se muestra en escala de grises. Tenga en cuenta las piscinas con forma de donut múltiples tinte destacando los espacios perisynaptic (flecha). </li></ol>

<ol>
	<li>Macleod, G. T., Marin, L., Charlton, M., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+Vesicles:+Test+for+a+role+in+presynaptic+Calcium+regulation.">Synaptic Vesicles: Test for a role in presynaptic Calcium regulation.</a> J. Neurosci. 24, 2496-2505 (2004).</li><li>Macleod, G. T., Hegstro, M., Wojtowicz, M., Charlton, M. P., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+Calcium+signals+in+Drosophila+motor+neuron+terminals.">Fast Calcium signals in Drosophila motor neuron terminals.</a> J. Neurophysiology. 88, 2659-2663 (2002).</li><li>Morales, M., Ferrus, A., Martinez-Padron, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+calcium-channel+currents+in+normal+and+csp+mutant+Drosophila+peptidergic+terminals.">Presynaptic calcium-channel currents in normal and csp mutant Drosophila peptidergic terminals.</a> Eur J Neurosci. 11, 1818-1826 (1999).</li><li>Stork, T., Engelen, D., Krudewig, A., Silies, M., Bainton, R. J., Kla&#168;mbt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+Function+of+the+Blood&#8211;Brain+Barrier+in+Drosophila.">Organization and Function of the Blood&#8211;Brain Barrier in Drosophila.</a> J. Neurosci. 28, 587-597 (2008).</li></ol>

Este procedimiento permite a largo plazo de imágenes en vivo de proteínas marcadas y los procesos celulares. La preparación en el tejido in situ que se describe tiene un sistema nervioso central intacto y funcionando, PNS y circuitos reflejos. Esta preparación de tejidos tiene ventajas sobre los protocolos estándar de larvas de mosca de los músculos, donde se estira el músculo de la pared del cuerpo larval (cuando se fija a cabo). El estiramiento puede distorsionar la morfología sináptica y de...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>HL-6: Artifical Drosophila hemolymph, with 5 mM L-glutamate added, and 2 mM Calcium.</td>
<td>Reagent</td>
<td>NA</td>
<td>NA</td>
<td>5 mM L-glutamate blocked muscle contractions. We used Molecular grade L-Glutamate (Sigma).
2 mM Calcium is close to physiological Calcium levels in natural larval hemolymph.
References: Macleod et al 2002 and Macleod 2004
</td>
</tr>
<tr>
<td>Dextran, Alex Fluor 680; 10,000 MW, anionic, fixable</td>
<td>Reagent</td>
<td>Molecular Probes/Invitrogen</td>
<td>D34680</td>
<td>Use a small volume perfusion chamber to keep the total volume of dye low</td>
</tr>
<tr>
<td>Anti-HRP-CY5 conjugate (goat)</td>
<td>Reagent</td>
<td>Jackson ImmunoResearc</td>
<td>123-175-021</td>
<td>Dilute 2.0 mg into 1 ml ddH2O; aliquot into 4 microliter aliquots. Freeze at &#x2013;20C. Dilute one aliquot into 100 microliters of HL-6</td>
</tr>
<tr>
<td>Alexa 647 antibody labeling kit</td>
<td>Reagent</td>
<td>Molecular Probes/Invitrogen</td>
<td>A10475</td>
<td>We prepared a total of 80 micro liters of conjugated primary antibody, and stored as 2 microliter aliquots. We diluted each aliquot into 100 microliter of HL-6 for labeling.</td>
</tr>
<tr>
<td>Custom Formulated Objective Oil, refractive index 1.3379</td>
<td>Reagent</td>
<td>Cargill Labs</td>
<td>Custom Formulated</td>
<td>&nbsp;</td>
<tr>
<td>Ultra Fine Forceps</td>
<td>Tool</td>
<td>Fine Science Tolls</td>
<td>11252-23 or 11295-20</td>
<td>&nbsp;</td>
<tr>
<td>Spring scissors</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>91500-09</td>
<td>&nbsp;</td>
<tr>
<td>Ultra fine clipper scissors</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>15200-00</td>
<td>&nbsp;</td>
<tr>
<td>Perfusion Chamber RC 20 Series</td>
<td>Tool</td>
<td>Warner Instruments</td>
<td>64-02222</td>
<td>&nbsp;</td>
<tr>
<td>Spinning Disc confocal</td>
<td>Microscope</td>
<td>Quorum</td>
<td>Quorum Wave FX</td>
<td>Mounted on a Leica DMI6000 Inverted Microscope</td>
</tr>		</tbody>
		</table>
		</div>

visualizing the live drosophila glial-neuromuscular junction with fluorescent dyes

Nuestro proyecto identificó GFP etiquetados estructuras gliales en la mosca en desarrollo larval sinapsis neuromuscular. Para ver el desarrollo de vida de la glía sinapsis nervio-músculo, hemos desarrollado una preparación de tejidos larvarios que tenían características de las larvas intactas en vivo, sino que también tenía buenas propiedades ópticas. Esta nueva preparación también se permitió el acceso de perfusates a la sinapsis. Hemos utilizado larvas de mosca, inmerso en la hemolinfa artificial, y se relajó sus contracciones rítmicas normales del cuerpo por el frío ellos. A continuación se diseca los segmentos posteriores de cada animal y con un alfiler de insectos contundente empujó la partes de la boca hacia atrás a través de la cavidad del cuerpo. Esta eversión de la pared cuerpo de la larva, como convertir un calcetín del revés. Hemos completado la disección con tijeras de disección ultra-fino y por lo tanto expuesto el lado visceral de los músculos de la pared del cuerpo. Las estructuras gliales en la UNM expresa GFP membrana dirigidos bajo el control de promotores específicos gliales. La membrana post-sináptica, la República Socialista Soviética (Reticula subsináptica) en el músculo expresado sinápticamente objetivo dsRed. Necesitábamos etiqueta agudamente la terminales de las neuronas motoras, la tercera parte de la sinapsis. Para ello se aplicaron anticuerpos primarios a HRP, conjugado con una emisión flurophore rojo lejano. Para probar las propiedades de la difusión de color en el espacio perisynaptic entre los terminales de las neuronas motoras y la SSR, se aplicó una solución de grandes moléculas de dextrano conjugado con flurophore emitiendo ahora de color rojo y las imágenes recogidas.

Watch this Scientific Journal Video about Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes at JoVE.com

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Se describen las características estructurales de la sinapsis neuromuscular Glia-en una nueva preparación de tejidos de adentro hacia afuera de las larvas de mosca en directo con tintes fluorescentes con microscopía confocal. Hemos marcado terminales de las neuronas en vivo con anticuerpos fluorescentes primaria de HRP, y también se visualiza el espacio perisynaptic con dextranos fluorescentes.

Visualizar el vivo Drosophila Gliales-unión neuromuscular con tintes fluorescentes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

visualizing-live-drosophila-glial-neuromuscular-junction-with

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Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Skeletal Muscle Relaxants

SCIENTISTS IN ACTION

10.7K vistas.  University of British Columbia - UBC. Se describen las características estructurales de la sinapsis neuromuscular Glia-en una nueva preparación de tejidos de adentro hacia afuera de las larvas de mosca en directo con tintes fluorescentes con microscopía confocal. Hemos marcado terminales de las neuronas en vivo con anticuerpos fluorescentes primaria de HRP, y también se visualiza el espacio perisynaptic con dextranos fluorescentes.

Video: Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Etiquetado de las células madre con tintes fluorescentes para la detección no invasiva con imágenes ópticas

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Investigación

Biología

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

La preparación de los Drosophila Los embriones para Live-Imaging utilizando el protocolo de gota

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

Marcaje fluorescente de Drosophila Estructuras del Corazón

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

Visualizar el corazón latiendo en Drosophila</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

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 ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Imágenes de Apoptotic Liquidación de células vivas durante<em&gt; Drosophila</em&gt; Embriogénesis

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

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 ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Medición simultánea de calcio mitocondrial y mitocondrial potencial de membrana en células vivas por microscopía fluorescente

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

Análisis de la transferencia dependiente de la unión Gap de miARN con 3D-FRAP Microscopía

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

Visualización y seguimiento de ARNm endógenos en cámaras de huevo Drosophila melanogaster en vivo

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...