このプロジェクトは、CIHRとNSERCによって資金を供給された。我々はDsRedのラベルが付いたSSR（BJライン）、およびUBCバイオイメージング施設を表すフライ株の創造に貢献するためのバーブJusiakに感謝します。

パート1：組織の準備<ol><li>私たちの目標は、神経系が損なわれていないハエの幼虫の組織の準備ですが、体壁筋の内側表面は人工の血リンパにさらされている、と近くに良い光学用顕微鏡のカバーガラスに配置することができます。言い換えれば、幼虫の準備裏返し。 従来の組織の準備が、カットピニングと体壁の筋肉をストレッチし、時には神経系の一部を除去を伴うので、私たちは異なるアプローチを必要としていました。 我々は構造的な特徴とシナプスの時間的構造変化をよく見て取得したいので、我々は、インサイドアウトの動物が欲しくて、私たちは神経系を維持するようにしたかった。我々はまた、組織をストレッチを避けるためにしたかった、と筋肉がけいれんし、私たちの画像を台無しにするようになるストレッチ受容体を、活性化する。 </li> <li>手始めに、動物を上演。第三幼虫を餌とサード幼虫をさまようことは我々がW3幼虫に説明していきますので、解剖に大きく、簡単にできます。この動物は、タビー表現型を有し、そして広いですので、裏返すことは容易である。 我々は、W3幼虫を含めて、我々は積極的にクロールを見たことが幼虫をのみを使​​用していました。 </li><li>蒸留水のペトリ皿の中で非常に柔らかいペイントブラシでウジの表面を清掃してください。クリーン幼虫は、より良い光学系を持ち、洗浄が細菌を減らします。 </li><li>約3mlの氷冷HL - 6、および人工血リンパの小さなペトリ皿に動物を譲渡する。動物が変化しない、と（約5分）リラックスするまで氷上に皿を置く。 </li><li>片手およびその他の春のはさみで非常に微細な先端の鉗子を保持する。私は私の支配的な手でハサミを使用してください。体壁を越え圧力をequlibrateにはさみが付いているボディの壁に小さな穴をあけます。 </li><li>皿の底を下に鉗子で軽く動物を持って、後部つのセグメントを切断。内臓を離れて摘出し、脂肪体は、おそらく、体腔外に移動します。また、この組織を切り取る。 </li><li>細かいピンセットでシャーレの底部に対して幼虫を保持する。 （鈍先端）の＃0昆虫ピンを持ち、幼虫の口の部分に対してそれを押す。裏返して、靴下を回っているような体腔を通じて口の部分を押してください。 </li><li>組織インサイドアウトでは、図のようになります：下記。超微細解剖ツールを使用して、体壁から脂肪体とtrachiolesを離れて分析する。本当にハードtrachiolesを引っ張ったり、神経系を切断しないようにしてください。 trachiolesをリッピングすると体壁の筋肉の穴をリッピングされます。 あなたができる限り多くの脂肪体を取り外します。これらの構造の両方は、あなたの組織の光学的品質を混乱させる。この準備を行う前にコーヒーをスキップしたいことがあります。 </li> <li>終了したら、筋肉は半透明、不透明または白ではないとなります。あなたがグルタミン酸なしHL - 6に組織の準備を置く場合は中枢神経系における運動パターンジェネレータが作業しているので、室温で、それは可能性がリズミカルに収縮したりします。 明らかに契約、または不規則に収​​縮体壁筋でプレップを使用することは避けてください。 体壁から無傷の内部が容易に背側と腹側の中心線に沿って半分に折るする傾向がある、準備が視覚化するために左または右の&quot;半動物&quot;を与えるように。 </li><li> 1.9などのワーナー室のような少量の商業室、、またはブリッジカバースリップの配置と顕微鏡用スライドのいずれかで組織をマウントします。取り付け組織についての提案は、パート3を参照してください。 </li></ol>パート2：HRPに対する蛍光標識一次抗体と神経ブートンラベリング。 <ol><li>ペトリ皿上にドロップでHL - 6でHRPに対する蛍光標識一次抗体の50マイクロリットルを入れて。染浴の裏返し体壁の準備を浸す。あなたは約5分後にニューロンの末端標識が見えますが、完全で明るいラベルのために、10〜20分間インキュベートすることがあります。 </li><li>室温でHL - 6の10〜30秒のために染料を洗い流す。非結合色素は、そのリンスサイクルは積極的である必要はなく、すぐにオフリンス。 </li><li>必要に応じて色素濃度とインキュベーション時間を変化させる。 </li></ol>パート3：デキストランflurophore共役とPerisynapticスペースラベリング<ol><li> HL - 6で希釈した蛍光共役デキストランの染料を希釈する。我々の目的のためによく働いた濃度であった。 </li><li>あなたがあなたの細胞外空間へのへの色素のアクセスのタイミングが心配されていない場合は、&quot;ブリッジ&quot;カバースリップ（パート4を参照）に染料の20マイクロリットル滴を入れて、染浴の準備を入金。 </li></ol>第4部：可視化のための組織を取り付け（共焦点顕微鏡による）。 あなたの準備を灌流する必要がない（短い観測）またはHL - 6小入浴のボリュームを保持する場合は、ダブルブリッジされたスライドのメソッドを使用しますあなたの準備を灌流する場合、灌流チャンバーを使用してみてください。我々は、MOを使用ワーナーの楽器からdified商工会議所。 両方のフォローのための詳細。 ダブルブリッジスライドに準備をマウント。 <ol><li>非常にきれいな顕微鏡スライドを使用してください。スライド上に瞬間接着剤二つの四角い、18ミリメートル（＃1.5）カバースリップ。カバースリップの端との間の2 mmのギャップを残す。 </li><li>接着剤が完全に乾燥させ、またはそれはあなたの準備の周りの水性媒体を介して奇妙なフィルムを形成します。 </li><li>カバースリップの端との間の幼虫インサイドアウトを置きます。幼虫が大きく、あなたの画像取得システムは、限られたピクセルの配列を持っている場合には、対角線上準備を配置する必要がある場合があります。 </li><li> 18ミリメートル＃1.5カバー、（非常にきれい）と組織をカバーしています。スライドは最小限のワセリンで、スリップに隣接する組織に貼付されて&quot;トップ&quot;カバースリップを付着する。 </li><li>上部カバースリップ上に1.3379の屈折率（あなたがHL - 6を使用している場合）とカーギルカスタムの目標の油の滴を入れて、このアセンブリを裏返しにします。 </li><li>目的に向かってあなたの顕微鏡、油側の組織のアセンブリを置き、そしてあなたの目的に焦点を当てる。 修正された灌流チャンバ内の組織の取り付け。 </li><li> RC 20室の床面を形成する接着剤1.5カバースリップ。 RC - 20の指示に従ってチャンバー内の組織を置きます。代わりに、チャンバーのための屋根を形成するためにカバースリップのNytexメッシュの一部を使用してください。 </li></ol>パート5：代表的な結果： <ol><li> インサイドアウトの組織の準備郭清のシーケンス： <img src="/files/ftp_upload/1154/fig1.jpg" alt="図1" /> W3幼虫、洗浄し（&quot;タビー&quot;表現型）（5.1.1）。 W3幼虫、解剖後2セグメント。あなたは、トランス体壁圧（矢印）で体腔から押し出さ脂肪体を見ることができます。解剖の前に体壁1分（5.1.2）で小さな穴を作ることによって内臓&quot;噴火&quot;を最小限に抑えるようにしてください。内臓と準備は裏返し準備（5.1.3）を回す前に解剖。準備（5.1.4）のルーメン内のピン（矢印）とほぼeverted準備、、。いくつかの脂肪体と接続されているtrachioles（5.1.5）で、筋肉が外側に今あり、キューティクルの内側にあります。ほぼすべての脂肪体と解剖trachioles（5.1.6）と完全に切除組織の準備。 <img src="/files/ftp_upload/1154/fig2a.jpg" alt="図2" width="600" height="200" /></li><li>幼虫のNMJで抗HRPを用いてラベリング住んでいます。グリア拡張子（アウト準備内側）と代表的なW3幼虫神経筋シナプス。運動ニューロンブートン端子はCy5との共役であるHRP（マゼンタ）、に対する一次抗体で標識されています。グリアのプロセスは、GFP（緑色）で標識されています。 <img src="/files/ftp_upload/1154/fig2b.jpg" alt="図3" /></li><li> Perisynapticスペースは蛍光準備裏返しでアレクサ680デキストランで視覚化。グリアプロセス（緑）が膜を対象とGFPで標識されている。筋肉の表面（青色）で、ポストシナプスSSRは、DsRedタンパク質で標識されている。アレクサデキストラン（赤）の形態では、細胞外の領域に集中している。デキストラン染料はperisynapticスペースでドーナツ型のプールを形成している。デキストランのラベリングおよびDsRed標識されたSSRはグレースケールで表示されます。 perisynapticスペース（矢印）を強調する複数のドーナツ状の色素プールを注意してください。 </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>

この手順では、ライブ標識タンパク質や細胞プロセスの長期的なイメージングを可能にします。私たちが説明の現場組織の準備はそのまま機能しCNS、PNSと反射回路を持っています。この組織の準備は、幼虫の体壁の筋肉が引き伸ばされている標準の幼虫のフライ筋肉プロトコル、（これが外部に固定されている場合）上の利点があります。ストレッチングシナプス形態を歪曲し、反射?...

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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

私たちのプロジェクトは、開発途上幼虫のフライ神経筋シナプスでGFP標識したグリアの構造を同定した。ライブグリア - 神経筋シナプスの発達を見て、私たちは生きて無傷の幼虫の特徴を持っていた幼虫の組織の準備を開発するだけでなく、良好な光学特性を持っていた。この新しい製剤は、シナプスへの灌流液のアクセスを可能にした。我々は、ハエの幼虫を用い、人工体液中のそれらを浸し、そして、それらを冷却することによって、正常なリズム体の収縮を緩和。次に、各動物の後部のセグメントをオフに切開し、鈍虫ピンで体腔を後方に口の部分をプッシュ。これは靴下裏返しにして回すように、幼虫体壁をeverted。我々は、超微細解剖鋏で解剖を完了したので、体壁筋の内臓側を露出。 NMJにおけるグリアの構造は、グリア特異的プロモーターの制御下にGFPをターゲット膜を表明した。シナプス後膜、筋肉のSSR（シナプス胞体）がシナプスDsRedを対象と表明した。我々は急性運動ニューロンの端末、シナプスの第三部にラベルを付ける必要がありました。これを行うために、我々は、遠赤色発光flurophoreに結合した、HRPに一次抗体を適用する。運動ニューロンの端末とSSRの間にperisynaptic空間に染料の拡散特性をテストするために、我々は、遠赤色発光flurophoreと収集した画像に結合した大規模なデキストラン分子の溶液を適用する。

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

我々は、共焦点顕微鏡で蛍光染料を使用して、ライブハエの幼虫の小説インサイドアウトの組織調製物におけるグリア - 神経筋シナプスの構造的特徴を説明した。我々は、HRPの蛍光灯の一次抗体とのライブニューロンの端末をラベル付けし、また蛍光デキストランでperisynapticスペースを可視化。

ライブの視覚化ショウジョウバエ</em蛍光色素で>グリア - 神経筋接合部

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

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

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Unit 1

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Skeletal Muscle Relaxants

SCIENTISTS IN ACTION

10.7K ビュー.  University of British Columbia - UBC. 我々は、共焦点顕微鏡で蛍光染料を使用して、ライブハエの幼虫の小説インサイドアウトの組織調製物におけるグリア - 神経筋シナプスの構造的特徴を説明した。我々は、HRPの蛍光灯の一次抗体とのライブニューロンの端末をラベル付けし、また蛍光デキストランでperisynapticスペースを可視化。

ビデオ: 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.

光イメージングによる非侵襲的検出のための蛍光色素で標識幹細胞

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 ...

生物学

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.

の調製ショウジョウバエ</emハンギングドロッププロトコルを使用してライブイメージングのための>胚

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.

の蛍光標識ショウジョウバエハートの構造

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.

の心臓の鼓動の可視化ショウジョウバエ</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.

蛍光顕微鏡を用いて線虫の中の細菌の可視化

 
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.

アポトーシス細胞のクリアランスのイメージング中に生きる<em&gt;ショウジョウバエ</em&gt;胚

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.

蛍光顕微鏡でストロミュール頻度を可視化

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.

蛍光顕微鏡による生細胞内のミトコンドリアのカルシウムとミトコンドリア膜電位の同時測定

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顕微鏡によるmiRNAのギャップジャンクション依存的転写の解析

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.

ライブショウジョウバエメラノガスター卵室における内因性mRNAの可視化と追跡

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

ライブの視覚化ショウジョウバエグリア - 神経筋接合部