Questo progetto è stato finanziato dalla CIHR e NSERC. Vorremmo riconoscere Jusiak Barb per contribuire alla creazione di ceppi volare esprimere DsRed etichettati SSR (linea BJ), e la Bio-imaging UBC Facility.

Parte 1: Preparazione del tessuto <ol><li> Il nostro obiettivo è una preparazione tessuto di larve di mosca in cui il sistema nervoso è intatto, ma la superficie interna del muscolo parete del corpo è esposto a una emolinfa artificiale, e può essere posizionato vicino ad un microscopio copertura antiscivolo per una buona ottica. In altre parole, un dentro e fuori la preparazione larvale. Poiché i preparativi dei tessuti convenzionali relativi al taglio, pinning e stretching il muscolo parete del corpo, ea volte di rimuovere parte del sistema nervoso, avevamo bisogno di un approccio diverso. Volevamo che gli animali inside-out perché volevamo ottenere una buona occhiata a caratteristiche strutturali e cambiamenti strutturali nel corso del tempo nella sinapsi, e abbiamo voluto mantenere il sistema nervoso intatto. Abbiamo anche voluto evitare l&#39;allungamento dei tessuti, e l&#39;attivazione di recettori, che renderebbe i muscoli a contrazione e rovinare le nostre immagini. </li> <li> Per cominciare, lo stadio gli animali. Alimentazione delle larve terzo e Wandering larve terzo sono grandi e facili da sezionare quindi dovremo dimostrare sul W3 larve. Questo animale si ha il fenotipo Tubby, ed è larga, quindi è facile da Evert. Abbiamo usato solo le larve che abbiamo visto attivamente strisciando, tra cui W3 larve. </li><li> Pulire la superficie di un verme con un pennello molto morbido in una capsula di Petri di H2O bidistillata. Pulire le larve hanno una migliore ottica e la pulizia riduce i batteri. </li><li> Trasferire l&#39;animale ad una piccola scatola di Petri di circa 3 ml di ghiaccio freddo HL-6, e emolinfa artificiale. Mettere il recipiente sul ghiaccio finché l&#39;animale smette di muoversi, e rilassa (circa 5 minuti). </li><li> Tenere pinze punta molto fine in una mano e le forbici primavera negli altri. Io uso le forbici con la mano dominante. Fai un piccolo foro nella parete del corpo con le forbici per equlibrate la pressione sulla parete del corpo. </li><li> Tenere l&#39;animale delicatamente con la pinza verso il basso sul fondo del piatto e tagliare il posteriore due segmenti. Sezionare via i visceri e il grasso corporeo probabilmente uscire dalla cavità del corpo. Tagliare questo tessuto anche. </li><li> Tenere le larve contro il fondo piatto con una pinza sottile. Tenere un pin # 0 insetto (con una punta smussata) e spingerlo contro le parti della bocca delle larve. Spingere le parti della bocca attraverso la cavità del corpo, come si sta girando un calzino dentro e fuori. </li><li> L&#39;esterno del tessuto sarà simile figura: al di sotto. Con ultra-sottile strumento di dissezione, sezionare via il grasso corporeo e trachioles dalla parete del corpo. Prova davvero difficile non tirare la trachioles, o disconnettere il sistema nervoso. Ripping il trachioles lacererà fori nella parete muscolare del corpo. Rimuovere il corpo il più grasso possibile. Entrambe queste strutture disturbare la qualità ottica del tessuto. Si consiglia di saltare il caffè prima di fare questa preparazione. </li> <li> Al termine il muscolo sarà traslucido, non opaco o bianco. Se si inserisce la preparazione dei tessuti in HL-6 senza glutammato, a temperatura ambiente, è probabile che si contrae ritmicamente perché i generatori di pattern motorio del sistema nervoso centrale stanno lavorando. Evitare di utilizzare prepara con il muscolo del corpo, ovviamente, contratta, o irregolarmente contratto muro. L&#39;interno intatto dal muro corpo tende facilmente a piegare a metà lungo le linee mediane dorsale e ventrale, quindi la preparazione dà un destro o sinistro &quot;semi-animale&quot; da visualizzare. </li><li> 1,9 Montare il tessuto sia in una camera di piccolo volume commerciale, come la camera di Warner, o un vetrino da microscopio con un accordo ponte slittamento di copertura. Vedere la parte 3 per suggerimenti sul tessuto di montaggio. </li></ol> Parte 2: Etichettatura Bouton neuronale con fluorescente anticorpo primario contro HRP. <ol><li> Mettere 50 litri di micro fluorescente primario anti-corpo contro HRP in HL-6 in una goccia su un piatto di Petri. Immergere il dentro-fuori di preparazione parete del corpo nel bagno di tintura. Si può vedere l&#39;etichettatura terminale del neurone dopo circa 5 minuti, ma incubare per 10-20 minuti, per un etichetta completa e luminoso. </li><li> Sciacquare il colorante per 10-30 secondi in HL-6 a temperatura ambiente. Non legato colorante sciacqua via in fretta, così il ciclo di risciacquo non è necessario essere aggressivi. </li><li> Variare la concentrazione di colorante e il tempo di incubazione, se necessario. </li></ol> Parte 3: etichettatura spazio Perisynaptic con destrano coniugato flurophore <ol><li> Diluire destrano colorante fluorescente coniugato diluito in HL-6. Una concentrazione che ha funzionato bene per i nostri scopi è stato: </li><li> Se non siete preoccupati per i tempi di accesso colorante fino al tuo spazio extracellulare, mettere una goccia 20 micro litri di colorante su un vetrino &quot;a ponte&quot; (vedi parte 4) e depositare la preparazione del bagno di tintura. </li></ol> Parte 4: Montaggio del tessuto per la visualizzazione (con microscopia confocale). Se non c&#39;è bisogno di perfusione la tua preparazione (osservazione breve) o se si vuole mantenere il volume delle acque di balneazione HL-6 piccoli, utilizzare il metodo della partita doppia scorrimento ponte Se vuoi perfusione tua preparazione, provare a utilizzare una camera di perfusione. Abbiamo utilizzato un moCamera solidificati Instruments Warner. Dettagli per entrambi seguono. Montaggio di preparazione su un doppio ponte di diapositive. <ol><li> Utilizzare un vetrino da microscopio molto pulito. Supercolla due quadrate, 18mm (# 1.5) coprioggetto sul vetrino. Lascia un gap 2 mm tra i bordi del coprioggetto. </li><li> Lasciate che la colla completamente asciutto, o si forma un film strano nel mezzo acquoso intorno alla vostra preparazione. </li><li> Posizionare il dentro e fuori le larve tra i bordi del coprioggetto. Potrebbe essere necessario posizionare la preparazione in diagonale, se le larve è grande e il sistema di acquisizione delle immagini è una serie limitata di pixel. </li><li> Coprire il tessuto con un coprioggetto # 1.5, 18mm (molto pulito). Rispettare il coprioggetto &quot;top&quot; della diapositiva apposto scivola tessuto di accompagnamento, con vaselina minimo. </li><li> Mettere una goccia di olio Cargill obiettivo personalizzati con un indice di rifrazione di 1,3379 (se si utilizza HL-6) sul vetrino superiore e capovolgere questa assemblea oltre. </li><li> Posizionare il gruppo del tessuto sul microscopio, lato del petrolio verso l&#39;obiettivo, e mettere a fuoco il tuo obiettivo. Montaggio del tessuto in una camera perfusione modificato. </li><li> Colla di 1,5 coprioggetto per formare un pavimento in camera di 20 RC. Posizionare il tessuto nella camera come da RC-20 istruzioni. Usare un pezzo di nytex maglia al posto di un vetrino per formare un tetto per la camera. </li></ol> Parte 5: Risultati Rappresentante: <ol><li> Inside-out tessuto preparazione sequenza dissezione: <img src="/files/ftp_upload/1154/fig1.jpg" alt="Figura 1" /> W3 larve, lavato (fenotipo &quot;Tubby&quot;) (5.1.1). W3 larve, 2 segmenti posteriore sezionato. Si può vedere il corpo di grasso spinto fuori dalla cavità del corpo da corpo di pressione trans-parete (freccia). Cercare di minimizzare viscerale &quot;eruzione&quot;, facendo un piccolo foro nella parete del corpo 1 minuto prima di dissezione (5.1.2). La preparazione con i visceri sezionato prima di accendere la preparazione dentro e fuori (5.1.3). La preparazione, per lo più estroflesso, con il perno (freccia) all&#39;interno del lume della preparazione (5.1.4). Il muscolo è ora sulla parte esterna e la cuticola è sulla parte interna, con un po di grasso corporeo e trachioles allegato (5.1.5). Una preparazione del tessuto completamente sezionato con quasi tutto il corpo grasso e trachioles sezionato (5.1.6). <img src="/files/ftp_upload/1154/fig2a.jpg" alt="figura 2" width="600" height="200" /></li><li> Vivere con l&#39;etichettatura anti-HRP al NMJ larvale. Un rappresentante W3 larvale sinapsi neuro-muscolare con estensione gliali (dentro e fuori-prep). Il motore neurone terminali bouton sono etichettati con un primario anti-corpo contro HRP (magenta), che è coniugato con Cy5. I processi gliali sono etichettati con GFP (verde). <img src="/files/ftp_upload/1154/fig2b.jpg" alt="figura 3" /></li><li> Spazio Perisynaptic fluorescenti visualizzati con Alexa 680 destrano in un dentro e fuori di preparazione. Il processo gliali (verde) è marcato con GFP membrana mirati. Il post-sinaptica SSR sulla superficie del muscolo (blu) è etichettato con DsRed. Il Alexa destrano (rosso) le forme si concentra nelle aree extracellulare. Il colorante destrano forme piscine a forma di ciambella negli spazi perisynaptic. L&#39;etichettatura destrano e DsRed SSR verranno indicati in scala di grigi. Si noti la serie di piscine a forma di ciambella colorante evidenziando gli spazi perisynaptic (freccia). </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>

Questa procedura permette di lungo termine imaging di vivere proteine ​​etichettati e processi cellulari. La preparazione in loco dei tessuti che abbiamo descritto ha un sistema nervoso centrale integro e funzionante, PNS e circuiti riflessi. Questa preparazione del tessuto ha dei vantaggi rispetto protocolli standard di volare larvale muscolare, in cui si distende il muscolo larvale parete del corpo (quando è appuntato fuori). Lo stretching può alterare la morfologia sinaptica e innescare riflessi contra...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<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

Il nostro progetto ha identificato GFP strutture etichettato gliali in via di sviluppo larvale volare sinapsi neuromuscolare. Per vedere lo sviluppo di vivere gliali-neuro-muscolare sinapsi, abbiamo sviluppato una preparazione larvale tessuto che aveva caratteristiche di larve vive intatto, ma ha avuto anche buone proprietà ottiche. Questo nuovo preparato ha anche consentito l&#39;accesso delle perfusates alle sinapsi. Abbiamo usato larve di mosca, immerso in emolinfa artificiale, e rilassato loro normali contrazioni ritmiche del corpo da loro agghiacciante. Poi abbiamo sezionato fuori i segmenti posteriori di ogni animale e con un perno insetti smussato spinto l&#39;apparato boccale indietro attraverso le cavità del corpo. Questo estroflesso il muro larvale corpo, come girare un calzino dentro-fuori. Abbiamo completato la dissezione con finissime forbici dissezione e quindi esposto il lato viscerale dei muscoli della parete del corpo. Le strutture gliali al NMJ espresso GFP mirati membrana sotto il controllo di specifici promotori gliali. La membrana post-sinaptica, la SSR (reticoli Subsynaptic) nei muscoli espresso sinapticamente mirati DsRed. Avevamo bisogno di etichettare acutamente i terminali dei motoneuroni, la terza parte della sinapsi. Per fare questo abbiamo applicato gli anticorpi primari di HRP, coniugata ad un far-rosso flurophore emissione. Per verificare le proprietà di diffusione colorante nello spazio perisynaptic tra i terminali del neurone motore e la SSR, abbiamo applicato una soluzione di grandi molecole destrano coniugato con estrema rosso flurophore emissione e le immagini raccolte.

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

Abbiamo descritto le caratteristiche strutturali del Glia-sinapsi neuromuscolare in un preparato romanzo Inside-out tessuto di larve di mosca dal vivo con coloranti fluorescenti con microscopia confocale. Siamo etichettati terminazioni neuronali vivere con anticorpi fluorescenti primario di HRP, e anche visualizzare lo spazio perisynaptic con destrani fluorescenti.

Visualizzazione di Live Drosophila Gliali-giunzione neuromuscolare con tinture fluorescenti

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

Skeletal Muscle Relaxants

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10.7K Views.  University of British Columbia - UBC. Abbiamo descritto le caratteristiche strutturali del Glia-sinapsi neuromuscolare in un preparato romanzo Inside-out tessuto di larve di mosca dal vivo con coloranti fluorescenti con microscopia confocale. Siamo etichettati terminazioni neuronali vivere con anticorpi fluorescenti primario di HRP, e anche visualizzare lo spazio perisynaptic con destrani fluorescenti.

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.

Etichettatura Cellule staminali con le tinture fluorescenti per la rilevazione non invasiva con 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 ...

Ricerca

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.

La preparazione di Drosophila Gli embrioni per Live-Imaging Utilizzando il protocollo goccia

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.

Etichette fluorescenti di Drosophila Cuore Strutture

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.

Visualizzare il cuore pulsante di 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.

Visualizzare batteri nei nematodi usando la microscopia a fluorescenza

 
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.

Vivere Imaging di apoptotica delle cellule libera nel corso<em&gt; Drosophila</em&gt; Embriogenesi

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.

Visualizzazione Frequenza Stromule con microscopia a fluorescenza

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.

Misurazione simultanea di mitocondriale calcio e mitocondriale potenziale di membrana in cellule vive di 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.

Analisi del trasferimento di gig Junction-dependent di miRNA con la microscopia 3D-FRAP

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.

Visualizzazione e monitoraggio di mRNA endogeni nelle camere uovo di Drosophila melanogaster dal vivo

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