この作品は、ワシントン大学にNIHの助成金DA018109とMH78823、およびNIH神経科学ブループリントコアグラントP30NS057105によってサポートされていました。

文化の準備<ol><li> 0から3の動物1生後からラットまたはマウスの海馬細胞の解離細胞の培養します。私たちの神経細胞はに番号0の厚さのカバーガラスに広がるコラーゲン層への付着を回す基本的なアストロサイト単分子層、に従っている。平方センチメートル当たり約500個の細胞の密度で神経細胞プレート。 </li><li>文化がシナプス発達と成熟を可能にするために10-14日のために成長することができます。さらにグリア成長を阻止するために抗有糸分裂AraCので試験管4日で文化を扱う。ニューロンの生存を高めるためにNeurobasal培地+ B27サプリメント半培地交換をin vitroで5日間でそれらを養う。 </li><li>培養期間中に、機能的に沈黙シナプスの数を変更するように設計された治療法を紹介。我々は、グルタミン酸シナプスの割合が大きい（〜80％）を黙らせるつの便利な方法は、30mMのカリウムと4時間の治療であることがわかります。弱い脱分極刺激によるインキュベーション時間が長いほど同じようなサイレン2を誘導する。 50μMフォルスコリン、アデニリルシクラーゼの活性化、4時間の刺激は、ベースライン3でサイレント端末の人口を目覚めさせるために使用することができます。 </li></ol>サイレントシナプスの可視化<ol><li>細胞は蛍光顕微鏡を用いて離散シナプス端末の可視化を容易にするためにin vitroで10-14に一日でうまく区切り神経突起のプロセスで、比較的低密度を持つ必要があります。 </li><li>蒸留水で5mMのFM1 -​​ 43FXのストック溶液を準備します。株価は4℃暗所で維持されるべきである℃のFM1 -​​ 43FXを使用してすべての実験はまた、暗所で行う必要があります。 </li><li> 100mM塩化ナトリウム、2mMの塩化カルシウム、1mMの塩化マグネシウム、10mMのグルコース、および10μMFM1 -​​ 43FXを含むHEPES緩衝生理食塩水で45 mMの塩化カリウムで2分間の文化に挑戦。このソリューションはまた、シナプス後受容体を遮断し、再発性のシグナル伝達を防ぐために、1μMNBQXと25μMのD - APVが含まれている必要があります。この課題は、エキソサイトーシスとエンドサイトーシスが可能なシナプス端子にラベルを付けます。チャレンジタイムと強度は、リサイクルのプールの4のすべての利用可能な小胞のエキソサイトーシスの少なくとも1ラウンドを引き起こすように設計されています。しかし、課題は、端末の追加のサイレンシングを引き起こすのに十分ではありません。 </li><li>同じ塩化カリウムなく、生理食塩水HEPES緩衝が、500μMAdvasep - 7との非特異的色素5を削除するために使用して、わずか3〜5秒のために文化を洗ってください。このステップのタイミングは、すべてのFM1 -​​ 43FXのラベルが失われますAdvasep - 7の長時間のアプリケーションとして重要であることに注意してください。 5つの2分間隔でAdvasep - 7なしで生理食塩水緩衝さHEPES内の次の洗浄。 </li><li> 10分間、4％パラホルムアルデヒドおよびPBS、pH7.4中0.2％グルタルアルデヒドで細胞を固定してください。一度簡単にPBSで細胞を洗浄し、次いでPBSで4％正常ヤギ血清、0.04％トリトンX - 100を含むブロッキング溶液で15分間反応させます。 FM1 -​​ 43FXもトリトンX - 100はここと後のステップで使用される量に非常に敏感であることに注意してください。一般的に言えば、FM1 -​​ 43FXのラベルは任意のトリトンX - 100がない場合に最適ですが、一部の洗剤には、その後の免疫染色のために、細胞を透過性が必要である。我々は0.04％のTriton X - 100は、シナプス前サイレントシナプスの検査のために最適化されている経験的に決定している。 </li><li> 1:2500の濃度でブロッキング溶液に一次vGluT - 1抗体を希釈します。室温で3時間vGluT - 1抗体希釈で穏やかに振盪/回転で細胞をインキュベートします。 FM1 -​​ 43FXの染料の漂白を防ぐために箔を使用して、培養皿を覆うようにしてください。 </li><li> PBSで2回細胞を洗浄してから、2つの汚れを区別するためにFM1 -​​ 43FXとは異なるスペクトル特性を持つ蛍光体に結合した抗モルモット抗体で細胞をインキュベートする。私たちの実験では、我々は、ブロッキング溶液で1:500でアレクサ555 -標識抗モルモット抗体を使用します。室温で30分間振とうしながら二次抗体で細胞をインキュベートする。 </li><li>細胞をPBSで4回洗浄しFluoromount - Gの小さなドロップを使用してスライドガラス上にカバースリップをマウントします。細胞はスライドが手前になるように向きをカバースリップにしてください。スライドが一晩乾燥することができます。 </li><li>我々は現在、アクティブと非アクティブなシナプスの画像を取得する準備が整いました。共焦点レーザー走査型顕微鏡1.4開口数で60 ×油の目標を準備します。カバースリップ側が客観的に直面していることを確認し、ステージ上でスライドを置きます。スライドを中心にした後、過度に密ではない神経突起のフィールドを探します。また、残留FM1 -​​ 43FXの染料として、細胞体の近くに撮影を避けるために推奨されていても十分に洗浄した後、そこに残ることができる。我々の実験の画像の場合、我々は一般的に51 51ミクロンのフィールドの大きさに拡大します。 </li><li>取得する27 0.3ミクロンの手順で7.8ミクロンの深さをカバーする特定のフィールドのZスタック。この範囲は、我々の文化の中で神経突起の厚さをカバーするのに十分です。 Z -スタック内の各ステップでは、顕微鏡の視野内のすべてのグルタミン酸作動性シナプスを識別するためvGluT - 1抗体の免疫蛍光励起レーザ光を最初のスキャン。同じフィールドは、FM1 -​​ 43FXの蛍光のための各段階で再スキャンされます。信号の飽和を回避しながら、与えられた実験のためのすべてをカバーグラスの間で一貫して中立密度のフィルタリングと光電子増倍ゲインを設定します。我々は通常、各実験の条件ごと≥5のフィールドを取得する。 </li><li>解析ソフトウェアを使用して、コンポジットシングルプレーンのイメージにZ -スタックを変換する。我々はこの変換に使用するプロセスは、各ピクセルでのあらゆる面からの最大の強度に基づいて画像を生成します。合成画像を閾値処理され、vGluT - 1の端子は、FM1 -​​ 43FX染色を参照することなく、分析のために識別されます。涙点と呼ばれるステンドグラスエリアは、、関心領域としてマークされます。我々は通常、フィールドごとに10涙点を選択します。次にFM1 -​​ 43FXのチャネルが閾値処理され、関心領域が重畳されています。解析ソフトウェアは、各シグナルの蛍光強度だけでなく、それぞれの涙の面積を定量化するために使用されます。絶対ピクセル基準または割合のピクセル基準は、非アクティブなシナプスからアクティブ区別するために使用することができます。 </li></ol>代表的な結果<ol><li>私たちの文化では、我々はvGluT - 1染色で定義されているグルタミン酸シナプスの70-80％は、検出可能なFM1 - 43染色2、6-8で示すことがわかります。共局在マーカーとのシナプス：緑FM1 -​​ 43蛍光と赤色vGluT - 1染色のオーバーレイ画像ではこれは黄色い涙点の豊富として反映されます。これらは、矢印で示されている。 </li><li> （赤）vGluT - 1陽性涙点の約20-30％がベースラインより上（緑）FM1 -​​ 43蛍光を持つように失敗します。これらは、非アクティブなシナプス前終末であり、例は、矢印で示されます。 </li><li>シナプスの第三の人口も観察されます。これらは、（赤）vGluT - 1染色のFM1 -​​ 43蛍光が欠けている（緑）のための肯定的です。これらは、アクティブなGABA作動性シナプスであり、例は、アスタリスクで示されます。これらは、我々の分析のほとんどから除外されますが、明示的vGluT - 1抗体の代わりに小胞性GABAトランスポーター抗体を用いて研究することができます。 </li></ol>

<ol>
	<li>Mennerick, S., Que, J., Benz, A., Zorumski, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+and+synaptic+properties+of+hippocampal+neurons+grown+in+microcultures+and+in+mass+cultures.">Passive and synaptic properties of hippocampal neurons grown in microcultures and in mass cultures.</a> J. Neurophysiol. 73, 320-332 (1995).</li><li>Moulder, K. L., Jiang, X., Taylor, A. A., Olney, J. W., Mennerick, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+activity+depresses+synaptic+function+through+an+effect+on+vesicle+priming.">Physiological activity depresses synaptic function through an effect on vesicle priming.</a> J. Neurosci. 26, 6618-6626 (2006).</li><li>Moulder, K. L., Jiang, X., Chang, C., Taylor, A. A., Benz, A. M., Conti, A. C., Muglia, L. J., Mennerick, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+specific+role+for+Ca2+-dependent+adenylyl+cyclases+in+recovery+from+adaptive+presynaptic+silencing.">A specific role for Ca2+-dependent adenylyl cyclases in recovery from adaptive presynaptic silencing.</a> J. Neurosci. 28, 5159-5168 (2008).</li><li>Sara, Y., Mozhayeva, M. G., Liu, X., Kavalali, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+vesicle+recycling+supports+neurotransmission+during+sustained+stimulation+at+hippocampal+synapses.">Fast vesicle recycling supports neurotransmission during sustained stimulation at hippocampal synapses.</a> J. Neurosci. 22, 1608-1617 (2002).</li><li>Kay, A. R., Alfonso, A., Alford, S., Cline, H. T., Holgado, A. M., Sakmann, B., Snitsarev, V. A., Stricker, T. P., Takahashi, M., Wu, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+synaptic+activity+in+intact+brain+and+slices+with+FM1-43+in+C.+elegans,+lamprey,+and+rat.">Imaging synaptic activity in intact brain and slices with FM1-43 in C. elegans, lamprey, and rat.</a> Neuron. 24, 809-817 (1999).</li><li>Rosenmund, C., Sigler, A., Augustin, I., Reim, K., Brose, N., Rhee, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+control+of+vesicle+priming+and+short-term+plasticity+by+Munc13+isoforms.">Differential control of vesicle priming and short-term plasticity by Munc13 isoforms.</a> Neuron. 33, 411-424 (2002).</li><li>Altrock, W. D., tom Dieck, S., Sokolov, M., Meyer, A. C., Sigler, A., Brakebusch, C., Fassler, R., Richter, K., Boeckers, T. M., Potschka, H., Brandt, C., Loscher, W., Grimberg, D., Dresbach, T., Hempelmann, A., Hassan, H., Balschun, D., Frey, J. U., Brandstatter, J. H., Garner, C. C., Rosenmund, C., Gundelfinger, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+inactivation+of+a+fraction+of+excitatory+synapses+in+mice+deficient+for+the+active+zone+protein+bassoon.">Functional inactivation of a fraction of excitatory synapses in mice deficient for the active zone protein bassoon.</a> Neuron. 37, 787-800 (2003).</li><li>Ting, J. T., Kelley, B. G., Lambert, T. J., Cook, D. G., Sullivan, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+precursor+protein+overexpression+depresses+excitatory+transmission+through+both+presynaptic+and+postsynaptic+mechanisms.">Amyloid precursor protein overexpression depresses excitatory transmission through both presynaptic and postsynaptic mechanisms.</a> Proc. Natl. Acad. Sci. USA. 104, 353-348 (2007).</li></ol>

意義<ol><li>一般的にシナプスは、測定可能な確率で送信機を解放することによって動作すると考えられている。自分や他人が仕事では、小胞、神経伝達物質のトランスポーターやその他の重要なシナプスのマーカー2、6-8のフル装備にもかかわらず、いくつかのシナプスは神経伝達物質を放出する難治性であることがはっきりする。我々は、ニューロンの基底のネットワーク活動が非...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>FM1-43FX</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>F-35355</td>
<td>A red-shifted FM4-64FX is also available, but has not proven as amenable to assays of presynaptic silencing.</td>
</tr>
<tr>
<td>Advasep-7</td>
<td>&nbsp;</td>
<td>CyDex</td>
<td>AR-0A7-001</td>
<td>&nbsp;</td>
<tr>
<td>vGluT-1</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>AB5905</td>
<td>&nbsp;</td>
<tr>
<td>Alexa 647-conjugated anti-GP</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A-21450</td>
<td>&nbsp;</td>
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<td>Fluoromount-G</td>
<td>&nbsp;</td>
<td>Southern Biotechnology Associates</td>
<td>0100-01</td>
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presynaptically silent synapses studied with light microscopy

シナプス可塑性の基礎となる学び、覚え、また神経毒性になることから特に有害な侮辱を防止する適応性を表す可能性があるために神経系の能力。それは神経伝達物質のグルタミン酸を含む小胞を放出するシナプス前終末の能力のデジタルスイッチングのオンとオフのように表されるので、我々は一部に興味深いものですシナプス可塑性の形態を研究している。ここでは、齧歯類の海馬から調製した解離細胞培養におけるシナプス前終末の活動状況を可視化するためのプロトコルを示す。方法は、一般的にシナプス小胞を標識するために使用されるスチリル色素FM1 -​​ 43の修正可能な形で染色を使用してアクティブなシナプスの検出に依存しています。この染色プロファイルは小胞性グルタミン酸輸送体1（vGluT - 1）に関係なく、活性化状態のすべてのグルタミン酸シナプスを標識するために設計された汚れに対する抗体と同じ端子の免疫染色と比較されます。私たちは、脱分極刺激はシナプス前サイレンシングを誘導することがわかります。ベースライン条件の下で静かであるシナプスの人口は、長期にわたる電気サイレンシングによってまたはcAMPシグナル伝達経路の活性化によって活性化することができます。

Watch this Scientific Journal Video about Presynaptically Silent Synapses Studied with Light Microscopy at JoVE.com

Presynaptically Silent Synapses Studied with Light Microscopy

グルタミン酸作動性シナプスはサイレントモードにアクティブモードを切り替えることができます。我々は齧歯類の神経細胞の分散培養でそのシナプス前の活動状況を示すと、アクティブなシナプスを可視化するためにFM1 -​​ 43色素の修正可能なフォームを使用し、すべてのグルタミン酸シナプスを可視化するvGluT - 1抗体による免疫染色可視化される。

光学顕微鏡で学ぶシナプス前サイレントシナプス

Synaptic plasticity likely underlies the nervous system's ability to learn and remember and may also represent an adaptability that prevents otherwise ...

presynaptically-silent-synapses-studied-with-light-microscopy

Research

JoVE Journal

Biology

11.4K ビュー.  Washington University School of Medicine. グルタミン酸作動性シナプスはサイレントモードにアクティブモードを切り替えることができます。我々は齧歯類の神経細胞の分散培養でそのシナプス前の活動状況を示すと、アクティブなシナプスを可視化するためにFM1 -​​ 43色素の修正可能なフォームを使用し、すべてのグルタミン酸シナプスを可視化するvGluT - 1抗体による免疫染色可視化される。...

ビデオ: Presynaptically Silent Synapses Studied with Light Microscopy

Preparation of Drosophila S2 cells for Light Microscopy

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos1, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells.
S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media2. In addition, they grow optimally and quickly at 21-24&deg;C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks.
Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA)3. S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is this: first, S2 cells (which can possess transgenes to express tagged markers) are treated by RNAi to eliminate a target protein(s). RNAi treatment time can be adjusted to allow for differences in protein turn-over kinetics and to minimize cell trauma/death if the target protein is important for viability. Next, the treated cells are transferred to a dish containing a coverslip pre-coated with conA to induce cells to spread and tightly adhere to the glass. Finally, cells are imaged with the researcher's choice of microscopy modes. S2 cells are particularly good for studies requiring extended visualization of live cells since these cells stay healthy at room temperature and normal atmosphere.

Preparation of Drosophila S2 cells for Light Microscopy

Drosophila Schneider (S2) cells are an increasingly popular system for the discovery and functional analysis of genes. Our goal is to describe some of the microscopic techniques that make S2 cells such an increasingly important experimental system.

の準備ショウジョウバエ</em光学顕微鏡用> S2細胞

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature ...

生物学

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

細胞の電気融合を蛍光顕微鏡で可視化

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

集積光と原子間力顕微鏡による研究機械的刺激に対する細胞応答を生きる

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

ターゲット-エステラーゼ誘起染料の読み込み（TED）とERカルシウムの直接イメージング

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Here, we present a method for light microscopy analysis of tracheal terminal cells in Drosophila larvae. This method allows for quick examination of branch and lumen morphology in whole animals and would be useful for analysis of individual mutants or screens for mutations affecting terminal cell development.

の検討<em&gt;ショウジョウバエ</em光学顕微鏡で&gt;幼虫気管ターミナルセル

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

多層はゼブラフィッシュの長期シート光顕微鏡用に取り付け

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

共焦点および多光子顕微鏡を用いて5蛍光タンパク質によってマーク造血幹細胞および前駆細胞の生体内でクローン追跡で

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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 ...

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid external development, tractable genetics, and translucency of the zebrafish, Danio rerio, LSFM has delivered insights into cardiac form and function at high spatial and temporal resolution without significant phototoxicity or photobleaching. Imaging of beating hearts challenges existing sample preparation and microscopy techniques. One needs to maintain a healthy sample in a constricted field of view and acquire ultrafast images to resolve the heartbeat. Here we describe optimized tools and solutions to study the zebrafish heart in vivo. We demonstrate the applications of bright transgenic lines for labeling the cardiac constituents and present novel gentle embedding and immobilization techniques that avoid developmental defects and changes in heart rate. We also propose a data acquisition and analysis pipeline adapted to cardiac imaging. The entire workflow presented here focuses on zebrafish embryonic heart imaging but can also be applied to various other samples and experiments.

We describe optimized tools to study the zebrafish heart in vivo with light sheet fluorescence microscopy. Specifically, we suggest bright cardiac transgenic lines and present new gentle embedding and immobilization techniques that avoid developmental and heart defects. A possible data acquisition and analysis pipeline adapted to cardiac imaging is also provided.