Este projeto foi financiado pelo CIHR e NSERC. Gostaríamos de agradecer Jusiak Barb para contribuir para a criação das cepas voar expressar dsRed SSR marcados (linha BJ), eo UBC Facility Bio-imagem.

Parte 1: Preparação do tecido <ol><li> Nosso objetivo é uma preparação de tecidos de larvas de moscas, onde o sistema nervoso está intacta, mas a superfície interior do músculo parede do corpo é exposto a uma hemolinfa artificial, e pode ser posicionada perto de uma lamínula de microscópio de ótica bom. Em outras palavras, uma de dentro para fora preparação larval. Desde preparações de tecido convencional envolvem corte, prendendo e alongamento do músculo parede do corpo e, por vezes retirando parte do sistema nervoso, precisávamos de uma abordagem diferente. Queríamos que os animais de dentro para fora, porque queríamos dar uma boa olhada em características estruturais e as mudanças estruturais ao longo do tempo na sinapse, e queríamos manter o sistema nervoso intacto. Nós também queríamos evitar esticar o tecido e ativando os receptores de estiramento, o que tornaria os músculos se contorcer e arruinar as nossas imagens. </li> <li> Para começar, estágio os animais. Alimentando larvas terceiro e Wandering larvas terceiro são grandes e fáceis de dissecar isso vamos demonstrar na W3 larvas. Este animal é tem o fenótipo Tubby, e é grande, por isso é fácil de Evert. Nós só usamos larvas que vimos ativamente rastejando, incluindo W3 larvas. </li><li> Limpe a superfície de um verme com um pincel muito macio em uma placa de Petri de bidestilada H2O. Larvas limpas têm melhor óptica e limpeza reduz bactérias. </li><li> Transferir o animal para uma pequena placa de Petri de cerca de 3 ml de gelo frio HL-6, e hemolinfa artificial. Coloque o prato sobre o gelo até que o animal pára de se mover, e relaxa (cerca de 5 minutos). </li><li> Segure uma pinça ponta muito fina em uma mão e uma tesoura de primavera no outro. Eu uso a tesoura com a mão dominante. Faça um pequeno buraco na parede do corpo com a tesoura para equlibrate a pressão do outro lado da parede do corpo. </li><li> Mantenha o animal gentilmente com a pinça para baixo no fundo do prato e cortar o posterior dois segmentos. Dissecar fora as vísceras e gordura corporal provavelmente vai sair da cavidade do corpo. Cortar esse tecido também. </li><li> Segure as larvas contra o fundo prato com uma pinça fina. Segure um alfinete # 0 inseto em (com uma ponta romba) e empurrá-lo contra a partes da boca das larvas. Empurre a partes da boca através da cavidade do corpo como se estivesse virando uma meia do avesso. </li><li> Dentro para fora do tecido será semelhante a figura: abaixo. Com ultra-fina ferramenta de dissecação, dissecar afastado a gordura corporal e trachioles da parede do corpo. Tente REALMENTE difícil não puxar o trachioles, ou desligar o sistema nervoso. Rasgando o trachioles vai rasgar buracos na parede muscular do corpo. Remover o corpo tanto quanto a gordura como você pode. Ambas as estruturas perturbar a qualidade óptica do seu tecido. Você pode querer pular o café antes de fazer essa preparação. </li> <li> Quando você terminar o músculo irá ser translúcido, não opaca ou branco. Se você colocar o tecido em prep HL-6 sem glutamato, à temperatura ambiente, ele provavelmente vai contrair ritmicamente, porque os geradores de padrão motor no SNC estão funcionando. Evitar o uso de preps com o músculo contraído obviamente, ou irregularmente contratados corpo parede. O interior intacto parede do corpo para fora prontamente tende a dobrar ao meio ao longo da linha média dorsal e ventral, de modo a preparação dá uma para a esquerda ou direita &quot;hemi-animal&quot; para visualizar. </li><li> 1,9 Monte do tecido em qualquer câmara de um pequeno volume comercial, tais como a câmara Warner, ou uma lâmina de microscópio com um arranjo de ponte tampa de deslizamento. Ver a parte 3 para sugestões sobre o tecido de montagem. </li></ol> Parte 2: Rotulagem Bouton Neuronal fluorescente etiquetado com anticorpo primário contra a HRP. <ol><li> Colocar 50 litros de micro fluorescente etiquetado primário anti-corpo contra a HRP em HL-6 em uma queda em uma placa de Petri. Mergulhe dentro para fora prep parede do corpo no banho de corante. Você pode ver neurônio rotulagem terminal após cerca de 5 minutos, mas incubar por 10-20 minutos, por uma etiqueta completa e brilhante. </li><li> Enxaguar o corante por 10-30 segundos na HL-6 em temperatura ambiente. Não-vinculados corante lavagens rapidamente, de modo que o ciclo de lavagem não precisa ser agressivo. </li><li> Variar a concentração de corante e tempo de incubação, conforme necessário. </li></ol> Parte 3: Rotulagem espaço Perisynaptic com dextran flurophore conjugado <ol><li> Diluir fluorescente conjugados dextran corante diluído em HL-6. Uma concentração que funcionou bem para os nossos propósitos foi: </li><li> Se você não está preocupado com o tempo de acesso ao corante em seu espaço extracelular, coloque uma gota 20 micro litro de corante em uma lamela &quot;ponte&quot; (ver parte 4) e depositar a preparação do banho de tintura. </li></ol> Parte 4: Montagem do tecido para visualização (com microscopia confocal). Se você não precisa perfundir sua preparação (observações curtas) ou você quer manter o volume de banho HL-6 pequenos, use o método de lâmina dupla em ponte Se você quiser perfundir sua preparação, tente usar uma câmara de perfusão. Usamos um moCâmara dified de Instrumentos Warner. Detalhes para os dois seguintes. Montagem de preparação em um slide duplo ponte. <ol><li> Use uma lâmina de microscópio muito limpo. Supercola dois quadrados, 18mm (# 1.5) lamínulas para o slide. Deixe um intervalo de 2 mm entre as bordas das lamínulas. </li><li> Deixe a cola secar completamente, ou ele vai formar uma película estranha sobre o meio aquoso em torno de sua preparação. </li><li> Posição dentro para fora larvas entre as bordas das lamínulas. Você pode precisar a posição da preparação em uma diagonal, se as larvas é grande e seu sistema de aquisição de imagem tem uma matriz de pixels limitada. </li><li> Cubra o tecido com uma lamela # 1.5, 18mm (muito limpo). Aderem a lamela &quot;top&quot; no slide afixada desliza tecido de acompanhamento, com vaselina mínimo. </li><li> Coloque uma gota de óleo Cargill objetivo personalizado com um índice de refração de 1,3379 (se você usa HL-6) sobre a lamela superior e virar esta assembléia acabou. </li><li> Posicione o conjunto de tecidos ao seu lado petróleo microscópio, em direção ao objetivo, e focar seu objetivo. Montagem do tecido em uma câmara de perfusão modificado. </li><li> Cola lamínula de 1,5 para formar um piso nos 20 RC câmara. Posicione o tecido na câmara como por RC-20 instruções. Use um pedaço de malha NYTEX em vez de uma lamela para formar um teto para a câmara. </li></ol> Parte 5: Resultados Representante: <ol><li> De dentro para fora do tecido seqüência dissecção preparação: <img src="/files/ftp_upload/1154/fig1.jpg" alt="Figura 1" /> W3 larvas, lavados (fenótipo &quot;Tubby&quot;) (5.1.1). W3 larvas, posterior 2 segmentos dissecados. Você pode ver de gordura corporal empurrado para fora da cavidade do corpo pelo corpo trans-tensão de parede (seta). Tentar minimizar &quot;erupção&quot; visceral, fazendo um pequeno buraco na parede do corpo um minuto antes da dissecação (5.1.2). A preparação com as vísceras, dissecados antes de virar a preparação de dentro para fora (5.1.3). A preparação, a maioria evertido, com o pino (seta) no interior do lúmen do prep (5.1.4). O músculo está agora do lado de fora ea cutícula é no interior, com alguns de gordura corporal e trachioles anexado (5.1.5). A preparação dos tecidos dissecados totalmente com quase todo o corpo de gordura e trachioles dissecados (5.1.6). <img src="/files/ftp_upload/1154/fig2a.jpg" alt="figura 2" width="600" height="200" /></li><li> Viver rotulagem usando anti-HRP no MNJ larval. Um representante W3 sinapse nervo-músculo larval com uma extensão de glial (de dentro para fora prep). O motor terminais neurônio bouton são rotulados com um primário anti-corpo contra a HRP (magenta), que é conjugado com Cy5. Os processos gliais são rotulados com GFP (verde). <img src="/files/ftp_upload/1154/fig2b.jpg" alt="figura 3" /></li><li> Espaço Perisynaptic fluorescente visualizado com Alexa 680 dextran em uma de dentro para fora prep. O processo glial (verde) é rotulado com GFP membrana alvo. O SSR pós-sinápticos na superfície muscular (azul) é rotulado com dsRed. O Alexa dextran (vermelho) formas concentra em áreas extracelular. O corante dextran formas piscinas em forma de anel nos espaços perisynaptic. A rotulagem dextran e dsRed SSR marcados são mostrados em escala de cinza. Observe a várias piscinas donut em forma de corante destacando os espaços perisynaptic (seta). </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 procedimento permite a longo prazo de imagens ao vivo de proteínas etiquetadas e processos celulares. O tecido em prep situ descrevemos tem um SNC intacto e em funcionamento, PNS e circuitos reflexos. Esta preparação do tecido tem vantagens sobre protocolos padrão voar larval muscular, onde o músculo da parede do corpo larval é esticada (quando ele é preso para fora). Alongamento pode distorcer a morfologia sináptica e desencadear reflexo contrações based. Nossa preparação foi dentro p...

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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

Nosso projeto identificou GFP rotulados estruturas glial no desenvolvimento larval sinapse neuromuscular voar. Olhar para o desenvolvimento de viver glial-nervo-músculo sinapses, desenvolvemos uma preparação do tecido larval que tinham características de larvas intactas ao vivo, mas também teve boas propriedades ópticas. Esta nova preparação também permitiu o acesso de perfusato para a sinapse. Usamos larvas de moscas, imersa-los em hemolinfa artificial, e relaxaram seus normais contrações rítmicas do corpo de arrefecimento-los. Em seguida, dissecados fora os segmentos posterior de cada animal e com um alfinete de inseto blunt empurrou a partes da boca para trás através da cavidade do corpo. Este evertido da parede do corpo larval, como transformar uma meia dentro para fora. Concluímos a dissecção com tesoura ultra-fina dissecação e, portanto, expostos ao lado visceral dos músculos da parede do corpo. As estruturas glial no MNJ expressa GFP membrana alvo sob o controle de promotores glial específico. A membrana pós-sináptica, a SSR (Reticula subsináptico) no músculo expressa sinapticamente alvo dsRed. Precisávamos aguda rótulo os terminais do neurônio motor, a terceira parte da sinapse. Para fazer isso, aplicamos anticorpos primários para HRP, conjugado a um flurophore emitindo far-vermelho. Para testar as propriedades de difusão de corante no espaço perisynaptic entre os terminais do neurônio motor e da SSR, aplicou-se uma solução de moléculas grandes Dextran conjugado com muito vermelho flurophore emissora e imagens recolhidas.

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

Descrevemos as características estruturais das sinapses Glia-neuromuscular em um romance de preparação dos tecidos dentro para fora-de larvas de moscas ao vivo usando corantes fluorescentes com microscopia confocal. Nós rotulados terminais de neurônios fluorescentes viver com anticorpos primários para HRP, e também visualizar o espaço perisynaptic com Dextranos fluorescente.

Visualizando o Live Drosophila Junção neuromuscular-Glial com corantes fluorescentes

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

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Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

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10.7K visualizações.  University of British Columbia - UBC. Descrevemos as características estruturais das sinapses Glia-neuromuscular em um romance de preparação dos tecidos dentro para fora-de larvas de moscas ao vivo usando corantes fluorescentes com microscopia confocal. Nós rotulados terminais de neurônios fluorescentes viver com anticorpos primários para HRP, e também visualizar o espaço perisynaptic com Dextranos fluorescente.

Vídeo: 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.

Rotulagem células-tronco com corantes fluorescentes para detecção não invasiva com imagens 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 ...

Biologia

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.

Elaboração de Drosophila Os embriões para Live-Imaging Usando o protocolo gota suspensa

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.

Labeling fluorescentes de Drosophila Estruturas Coração

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 o coração batendo no 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.

Visualizando bactérias em nematóides por microscopia de fluorescência

 
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.

Imagens ao vivo de Apuramento celular por apoptose durante<em&gt; Drosophila</em&gt; Embriogênese

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.

Visualizando Frequency Stromule com microscopia de fluorescência

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.

Medição simultânea de mitocondrial de cálcio e mitocondrial potencial de membrana em células vivas por Microscopia 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.

Análise do Gap Junction-dependente Transferência de miARN com 3D-FRAP Microscopia

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.

Visualizando e rastreando mRNAs endógenas em câmaras de ovo de Drosophila melanogaster ao vivo

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