Bu proje CIHR ve NSERC tarafından finanse edildi. Barb Jusiak dsRed etiketli SSR (BJ çizgi) ve UBC Biyo-görüntüleme Tesis ifade sinek suşlarının yaratılmasına katkıda bulunmak için kabul etmek istiyorum.

Bölüm 1: Doku Hazırlama <ol><li> Amacımız, sinir sisteminin sağlam olduğunu sinek larvaları bir doku hazırlama, ancak vücut duvarı kasının iç yüzeyi yapay bir hemolimf maruz kalan ve iyi optik mikroskop kapağı kayma yakın yerleştirilmiş olabilir. Diğer bir deyişle, larva hazırlık çıkış içinde. Kesme, iğneleme ve vücut duvarı kas germe ve bazen sinir sisteminin bir parçası kaldırarak geleneksel doku preparatları içeren bu yana, biz farklı bir yaklaşım gerekiyordu. Biz, yapısal özellikleri ve sinaps zaman içinde yapısal değişiklikler iyi bir görünüm almak istedim, çünkü iç-dış hayvanlar istedim, ve sinir sistemi sağlam tutmak istedim. Ayrıca doku germe önlemek istedim ve kasları kasılan ve görüntü berbat gerilme reseptörleri aktive. </li> <li> Yeni başlayanlar için, hayvanların sahne. Üçüncü larva Beslenme ve üçüncü larva Wandering W3 larvaları açılayacağız incelemek için büyük ve kolay. Bu hayvan Tubby fenotip ve geniş, bu yüzden tersyüz etmek kolaydır. Biz sadece biz aktif W3 larvaları da dahil olmak üzere, tarama gördüğünü larvaları kullanılır. </li><li> Bir kurtçuk çift distile H2O bir petri çok yumuşak bir fırça ile yüzeyi temizleyin. Temizlik larvalar daha iyi optik ve temizleme bakterileri azaltır. </li><li> HL-6, 3 ml buz hakkında küçük petri ve yapay hemolimf hayvan aktarın. Hayvan hareket durur ve (yaklaşık 5 dakika) rahatlatır kadar buz üzerinde tabak koyun. </li><li> Bir yandan ve diğer yaylı makas çok ince uçlu forseps tutun. Benim dominant elin makas kullanın. Vücut duvarı boyunca basınç equlibrate makas ile vücut duvarında küçük bir delik açın. </li><li> Hayvan aşağı çanak alt forseps ile yavaşça tutun ve arka iki kesimi kesmek. Organlar uzak inceleyin ve yağ vücut olasılıkla vücut boşluğunda hareket edecek. Bu doku da kesip. </li><li> Larvaları ince forseps ile çanak alt karşı tutun. # 0 böcek pin tutun (künt bir ucu ile) ve larvaların ağız parçaları karşı itin. Eğer dışarı içinde bir çorap dönüm gibi vücut boşluğu ile ağız parçaları itin. </li><li> Doku içinde dışarı rakam gibi görünecektir: aşağıda. Ultra-ince kesme aleti ile yağ vücut ve trachioles vücut duvarından uzak teşrih. Trachioles çekmek için gerçekten zor değil deneyin, ya da sinir sistemi çıkarın. Trachioles Kopyalamak vücut duvarı kas delik rip. Mümkün olduğu kadar yağlı vücut çıkarın. Bu yapıların her ikisi de doku optik kalitesi bozabilir. Bu preparatın yapmadan önce kahve atlamak isteyebilirsiniz. </li> <li> Bitirdiğinizde kas şeffaf, opak veya beyaz olacaktır. Glutamat olmadan doku hazırlık HL-6 içine koyarsanız, MSS, motor desen jeneratörler çalışma olduğu için, oda sıcaklığında, büyük olasılıkla ritmik sözleşme olacaktır. Açıkça sözleşmeli ya da düzensiz sözleşmeli vücut duvarı kas preparatlarıyla kullanmaktan kaçının. Vücut duvarından dışarı bozulmamış içinde kolayca dorsal ve ventral midlines boyunca yarım kat eğilimindedir, bu nedenle hazırlık görselleştirmek için sol veya sağ hemi-hayvan &quot;verir. </li><li> 1.9 Warner odası gibi küçük hacimli ticaret odasının, ya da Köprülü kapak kayma düzenleme ile bir mikroskop lamı ya doku monte edin. Montaj doku önerileri için bölüm 3 bakın. </li></ol> Bölüm 2: HRP karşı floresan etiketli primer antikor ile Nöronal Bouton Etiketleme. <ol><li> HL-6 HRP karşı floresan etiketli birincil anti-beden 50 mikro litre petri bir damla koyun. Iç-dış boya banyosu içinde vücut duvarı hazırlık bırakın. Yaklaşık 5 dakika sonra nöron terminaline etiketleme, ancak tam ve parlak bir etiket için, 10-20 dakika boyunca inkübe. </li><li> HL-6 oda sıcaklığında 10-30 saniye boya durulayın. Sigara bağlı boya durulama agresif olması gerekmez, çabuk durular. </li><li> Boya konsantrasyonu ve inkübasyon süresi gerektiği gibi değişir. </li></ol> Bölüm 3: dekstran flurophore konjuge Perisynaptic alan etiketleme <ol><li> Sulandırınız floresan konjuge dekstran boya HL-6 seyreltilir. Iyi amaçlar için çalışan bir konsantrasyon: </li><li> Ekstrasellüler alanı içine boya erişim zamanlaması hakkında söz konusu değilse, &quot;köprülenmiş&quot; lamel (bkz. bölüm 4), boya, 20 mikro litre damla koyun ve boya banyosunda hazırlık yatırmak. </li></ol> Bölüm 4: görselleştirme (konfokal mikroskobu ile) için doku Montajı. (Kısa gözlemler) hazırlık serpmek gerek yok veya HL-6 küçük banyo hacmi tutmak istiyorsanız, çift köprülenmiş slayt yöntemi kullanın Hazırlık serpmek istiyorsanız, bir perfüzyon odasında kullanmayı deneyin. Biz bir moWarner Instruments dified Odası. Hem de takip Ayrıntılar. Montaj bir çift köprülenmiş slayt hazırlık. <ol><li> Çok temiz bir mikroskop lamı kullanın. Superglue slayt üzerine iki kare, 18mm (1.5) lamelleri. Lamelleri kenarları 2 mm arasında bir boşluk bırakın. </li><li> Tutkal tamamen kurumasına izin verin, ya da hazırlık etrafında sulu ortam içinde garip bir film tabakası oluşturur. </li><li> Lamelleri kenarları arasındaki larvaları dışarı içine yerleştirin. Larvaların büyük ve görüntü toplama sistemi sınırlı bir piksel varsa diyagonal bir hazırlık konumuna gerekebilir. </li><li> 18mm # 1.5 lamel, (çok temiz) ile doku örtün. Slayt, minimal vazelin, doku sınırdaş fişleri yapıştırılmış &quot;top&quot; lamel uyun. </li><li> Üst lamel 1,3379 bir kırılma indeksi (HL-6) ile Cargill özel amacı petrol bir damla koyun ve bu derleme ters çevirin. </li><li> Hedefi doğru mikroskop, petrol yan doku montaj pozisyonu ve objektif odak. Değiştirilmiş bir perfüzyon odasında doku Montajı. </li><li> 20 RC odasında bir zemin oluşturmak için Tutkal 1.5 lamel. RC-20 talimatlara göre odasında doku yerleştirin. Odası için bir çatı oluşturmak yerine bir lamel Nytex mesh bir parça kullanın. </li></ol> Bölüm 5: Temsilcilik Sonuçları: <ol><li> Tersyüz doku hazırlanması diseksiyonu sırası: <img src="/files/ftp_upload/1154/fig1.jpg" alt="Şekil 1" /> W3 larvaları, yıkanmış (&quot;Tubby&quot; fenotipi) (5.1.1). W3 larvaları, arka 2 segmentleri disseke. , Trans-vücut duvarı basıncı (ok) ile vücut boşluğu dışına itti yağ vücut görebilirsiniz. Visseral &quot;patlaması&quot; diseksiyonu önce vücut duvarı 1 dakika (5.1.2) küçük bir delik yaparak en aza indirmeye çalışın. Organları ile hazırlık dışarı içinde hazırlık (5.1.3) açmadan önce disseke. Pin (ok) (5.1.4) hazırlık lümen içinde çoğunlukla dışa dönük hazırlık,. Kas dışında ve manikür, (5.1.5) bağlı bazı yağlı vücut ve trachioles, iç. Neredeyse tüm yağlı vücut ve disseke trachioles (5.1.6) ile tam olarak disseke doku hazırlanması. <img src="/files/ftp_upload/1154/fig2a.jpg" alt="Şekil 2" width="600" height="200" /></li><li> Canlı larva NMJ anti-HRP kullanarak etiketleme. Glial bir uzantısı (dışarı hazırlık içinde) bir temsilci W3 larva sinir-kas sinaps. Cy5 için konjuge HRP (magenta), karşı bir anti-vücudun birincil motor nöron bouton terminalleri ile etiketlenir. Glial süreçleri GFP (yeşil) ile etiketlenir. <img src="/files/ftp_upload/1154/fig2b.jpg" alt="Şekil 3" /></li><li> Perisynaptic alan floresan hazırlık dışında bir iç Alexa 680 dekstran ile görüntülendi. Glial süreci (yeşil), membran hedef GFP ile etiketlenir. Kas yüzeyinde post-sinaptik SSR (mavi) dsRed etiketlenir. Alexa dekstran (kırmızı) formları ekstrasellüler alanlarda yoğunlaşmaktadır. Dekstran boya perisynaptic alanlarda çörek şeklinde havuzlar oluşturur. Dekstran etiketleme ve dsRed etiketli SSR gri tonlamalı gösterilmiştir. Perisynaptic alanlarda (ok) vurgulayarak çok çörek şeklinde boya havuzları unutmayın. </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>

Bu prosedür, canlı etiketli proteinler ve hücre süreçlerinin uzun vadeli görüntüleme izin verir. Açıklanan in situ doku hazırlık ve işleyişi sağlam bir merkezi sinir sistemi, PNS ve refleks devreler vardır. Bu doku hazırlık larva vücut duvarı kas gergin standart larva, sinek kas protokolleri, (tutturulmuş.) Üzerinde avantajları vardır. Esneme sinaptik morfolojisi deforme ve refleks tabanlı kasılmaları tetikleyebilir. Hazırlık dışarı içinde mekanik olarak dayanıklı ve gerçek z...

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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

Projemiz, gelişmekte olan larva, sinek nöromüsküler sinaps GFP etiketli glial yapıları belirledi. Canlı glial-sinir-kas sinaps geliştirme bakmak için canlı bozulmamış larvalarının özellikleri olan bir larva doku hazırlama geliştirdi, ama aynı zamanda iyi bir optik özellikleri vardı. Bu yeni hazırlık sinaps guruplarına erişim için izin verdi. Biz, sinek larvaları kullanılan yapay hemolimf onları dalmış ve onları ürpertici normal ritmik vücut kasılmaları rahat. Sonra her bir hayvanın arka kesimlerine disseke ve künt bir böcek iğne ile vücut boşluğu yoluyla ağız parça geriye itti. Bu çorap, iç-dış torna gibi, larva vücut duvarının dışa dönük. Biz ultra-ince diseksiyon diseksiyon makas ile tamamladı ve böylece vücut duvarı kasları visseral yan maruz. NMJ glial yapıları glial özel yararlanıcı kontrolü altında membran hedeflenen GFP dile getirdi. Kas post-sinaptik membran, SSR (Subsynaptic Reticula) synaptically dsRed hedef dile getirdi. Biz akut motor nöron terminalleri, sinaps üçüncü bölümü etiket gerekiyordu. Bunu yapmak için çok kırmızı yayan flurophore konjuge primer antikor, HRP uygulanır. Motor nöron terminalleri ve SSR arasında perisynaptic uzaya boya difüzyon özellikleri test etmek için, uzak-kırmızı yayan flurophore ve toplanan görüntüleri konjuge büyük Dextran&#39;ın moleküllerin bir çözüm uyguladı.

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

Biz sinek larvaları konfokal mikroskobu ile floresan boyalar kullanarak canlı bir roman Tersyüz doku hazırlanması Glia nöromüsküler sinapsların yapısal özellikleri nitelendirdi. Biz HRP için flüoresan primer antikor ile canlı nöron terminalleri etiketli ve floresan Dextrans perisynaptic alan görüntülendi.

Canlı görselleştirme Drosophila</emFloresan Boyalar> Glial nöromüsküler Kavşağı

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 Görüntüleme.  University of British Columbia - UBC. Biz sinek larvaları konfokal mikroskobu ile floresan boyalar kullanarak canlı bir roman Tersyüz doku hazırlanması Glia nöromüsküler sinapsların yapısal özellikleri nitelendirdi. Biz HRP için flüoresan primer antikor ile canlı nöron terminalleri etiketli ve floresan Dextrans perisynaptic alan görüntülendi.

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.

Optik Görüntüleme ile non-invaziv Algılama için Floresan Boyalar Kök Hücreler Etiketleme

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 ...

Biyoloji

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.

Hazırlanması Drosophila</emAsma Bırak Protokolü kullanarak Canlı Görüntüleme> Embriyolar

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.

VE Floresan Etiketleme Drosophila Kalp Yapıları

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.

Dayak Kalp görselleştirme 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.

Floresan Mikroskobu kullanılarak Nematodlar Bakteriler görselleştirme

 
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.

Apoptotik hücresi Temizliği görüntülenmesi sırasında Canlı<em&gt; Drosophila</em&gt; Embriyo

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.

Floresan Mikroskopi ile Stromule Frekansı görselleştirme

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.

Floresan Mikroskopi Canlı hücreler Potansiyel Mitokondriyal Kalsiyum ve Mitokondriyal Membran Eşzamanlı Ölçümü

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.

3D-FRAP Mikroskopisi ile Gap Birleşimine Bağlı MiRNA Transferinin Analizi

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.

Canlı Drosophila melanogaster yumurta Chambers Içinde endojen mrnas görselleştirme ve izleme

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

Canlı görselleştirme Drosophila Glial nöromüsküler Kavşağı