我々は、入力とパディーヤラボのメンバーからのコメントに感謝の意を伝えたい。この作品で線虫株は研究資源のためのNIHナショナルセンター（NCRR）によって運営されている線虫の遺伝学センターから提供された。我々は、共焦点顕微鏡について技術的な支援のために博士ロンターンブルを認め、感謝しています。この作品は、PAPに国立科学財団（NSF-IOS、キャリア）からの助成金によって支えられてきた

1。試料調製<ol><li>適切なトランスジェニックCを生成または取得elegansはトランスジェニックの手法を用いて、関心のあるまたは線虫遺伝ストックセンター（CGC）や同僚への負担が少なくなります。細胞分裂のマーカーとして染色体や中心体を視覚化するために5;このケースでは、（PIE-1 :: GFP ::パイH2B-1 :: TBG-1 :: GFP）株TH32を使用しています。 </li><li> 3つの方法のいずれかを使用して同期化された集団を生成する：1）次亜塩素酸溶液中で妊娠成人を崩壊させ、残りの胚6を使用して 、2）播種板上に妊娠成人をピックアップし、それらを1〜2時間のために胚を置く、または3）L4を選ぶことができ混合集団とmoveから新しいシードプレートに幼虫。 </li><li>孵化から約96時間かかります妊娠青年期、〜20℃、L1幼虫または24時間後、L4の脱皮から72時間で線虫を育てています。 子宮内で胚（2-20セル）IMAGに最適な大割球と核を含むるサブ携帯とサブ核の変化であった。この方法論は、初期胚の豊富な数を含む若年成人の集団を生成する手段を提供します。 </li></ol> 2。スライドの調製と酸素欠乏症商工会議所は、設定<ol><li>十分なサイズの蓋で覆われた大きなシャーレまたはビンの内側に湿らせたペーパータオルを配置することによって準備されたスライドを保持するために、湿潤チャンバーを作る。 </li><li>マイクロ波あるいは水浴を介してガラスに混合物を加熱することにより脱イオンH 2 Oで溶融2％アガロースを準備します。 </li><li>きれいなガラス顕微鏡スライド上に暖かいアガロースの1-3滴を置きます。直ちにアガロースドロップ（複数可）の上に垂直に反転した2枚目のスライドを配置し、その結果、パッドが薄いと気泡の自由になるように少し押します。 </li><li>クールゆっくりとスライドの1つ上のアガロースパッドはそのままにして2顕微鏡スライドを分解する時間を確保します。アガロースパッドがinterfeないように十分に薄いようにすることが重要です後で顕微鏡を集中する能力を使用して再。サンプルを視覚化する能力はまた、顕微鏡間で異なることができ、使用中の特定の目的の光学距離に依存する。 </li><li>小さな正方形にアガロースパッドを形成するためにクリーンなかみそりの刃を使用しています。使用する準備ができるまで準備湿潤チャンバーに保管してください。 </li><li>慎重に、同じクリーンなカミソリの刃を用いて、アガロースパッドの端を取るとラウンド25ミリメートルマイクロカバーガラス上にスライドからそれを転送し、パッドの下に気泡の蓄積を回避する。使用するために選択した丸いカバーガラスはmicroincubatorで適切にフィットします。 </li><li>麻酔薬として使用するためのアガロースパッドに麻酔薬（0.5％tricaine、M9のバッファ内の0.05％tetramisole）のドロップを追加します。麻酔薬のドロップに5月10日ワームや場所を選ぶ。動きを止めるためにワームの1から3分を許可します。 </li><li>成人子宮内の胎児を観察するための理論的根拠はなく、解剖した胚は、胚desiccaの可能性を最小限にすることですると胚全体で窒素ガスの流れによる移動。 </li><li>すぐにガスがチャンバを通して流されるように脱水からワームを防ぐために、ワームの上にハロカーボンオイルを一滴を追加します。ハロカーボン油は粘性があるので、一つはワームワームにオイル·アンド·ドロップを収集するために選ぶのピペットチップまたはチップを使用することができます。 </li><li>一度ワームを含む円形カバーガラスの準備が整ったので、ライデン閉鎖灌流microincubatorの二つの部分を分離し、カバーガラスを慎重にボトムリングに直立して配置されます。チャンバーの両側は、その後しっかりと一緒に閉鎖されています。 </li><li>チャンバーはその後、柔軟なプラスチックチューブを介して窒素ガスをタンクに接続されており、顕微鏡ステージ（ 図1C）の適当な位置に配置されます。この時チューブとmicroincubatorは慎重にチューブのチャンバの側面に沿ってポートプラグの確実な取り付けを確実にすることによって、任意の漏れがないかチェックする必要があります。一つは、ALができますとても軽くリークが存在するかどう形成する気泡を探すためにチューブを介してSOAP少量の水を広める。 </li></ol> 3。酸素欠乏症で顕微鏡<ol><li>低倍率で動物を見つけ、その後、そのような大人の胚割球や卵母細胞のような関心の画像組織または細胞に適した高倍率（このアプリケーションの64倍）に移動します。 GFPシグナルを表示し、目的の細胞（s）のGFP融合タンパク質を見つけるために488nmのレーザーをオンにします。 </li><li>細胞周期停止の特定の段階で逮捕を可視化するために（ 例えば、前期の後期）細胞分裂の前段階（ 例えば後期中間期または早期前期）で割球を識別します。このような中心小体の位置、染色体凝縮の開始、染色体の場所と核膜の有無などの細胞マーカーは、細胞周期段階（ 図2A）を識別するのに役立ちます。 </li><li>チャンバーは、窒素ガス（99.999％で灌流さN 2、&lt;2 ppmのO 2）。我々は6 psiの圧力を使用しかし、この場合には圧力が各microincubatorのサイズと、使用中のチューブの直径に応じて調整し、最適化する必要があるかもしれません。窒素は、必要に応じて焦点面を調整し、チャンバーを埋めるように割球（s）を継続的に監視します。 </li><li>この時点で、タイムラプスイメージングが始まりました。イメージングは​​、核の内膜に染色体のこの場合前期の逮捕とドッキングで、興味のある現象を捕捉するために必要な限りのために行われている。画像は30分を超えないために10秒ごとに一度取られます。画像キャプチャとゲインやレーザパワー等の設定の周波数が必要に応じて調整されるべきである。 </li><li>胚は無酸素環境に対応する時間は無酸素暴露時に細胞周期の段階に応じて変えることができる。一般的に酸素欠乏によって誘導された前期の逮捕は、窒素ガス流を開始してから20〜30分以内に起こる。我々は、酸素欠乏によって誘導された細胞周期停止occuを観察している&lt;20分でRRING。画像はこの時間枠の間に取得することができる、または特定の画像は後でタイムラプスシリーズから単離することができる。追加のオプションは、胚で細胞内構造を可視化するビデオ顕微鏡を使用することです。 </li><li>酸素欠乏によって誘導された逮捕された状態からの回復を文書化するために、ガスがオフになっていて、チャンバーをタイムラプスシリーズを記録しながら、拡散によって酸素正常状態に戻ることを許可されています。染色体の細胞周期の進行およびドッキング解除の再開は、典型的には窒素ガスをオフにして5〜20分以内に起こる。 </li><li>別の実験では無酸素インジケーター、レサズリンは、フロースルーマイクロチャンバ内に配置されたことに注意してください、それは窒素ガスフローが開始された後チャンバーは約19分の平均で無酸素になることを決定した。 </li><li>画像と動画は、Imaris、イメージJまたはPhotoshopを使用して処理し、ディスプレイ用のQuickTimeにインポートされます。 </li></ol>

<ol>
	<li>Powell-Coffman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+signaling+and+resistance+in+C.+elegans.">Hypoxia signaling and resistance in C. elegans.</a> Trends Endocrinol. Metab. 21 (10), 435-440 (2010).</li><li>Padilla, P. A., Ladage, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suspended+animation,+diapause+and+quiescence:+Arresting+the+cell+cycle+in+C.+elegans.">Suspended animation, diapause and quiescence: Arresting the cell cycle in C. elegans.</a> Cell Cycle. 11, (2012).</li><li>Hu, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dauer.">Dauer.</a> WormBook. , 1-19 (2007).</li><li>Zhou, K. I., Pincus, Z., Slack, F. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Nematode Caenorhabditis elegans. , 587-606 (1988).</li><li>Padilla, P. A., Nystul, T. G., Zager, R. A., Johnson, A. C., Roth, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dephosphorylation+of+Cell+Cycle-regulated+Proteins+Correlates+with+Anoxia-induced+Suspended+Animation+in+Caenorhabditis+elegans.">Dephosphorylation of Cell Cycle-regulated Proteins Correlates with Anoxia-induced Suspended Animation in Caenorhabditis elegans.</a> Mol. Biol Cell. 13, 1473-1483 (2002).</li><li>Hajeri, V. A., Trejo, J., Padilla, P. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suspended+animation+in+C.+elegans+requires+the+spindle+checkpoint.">Suspended animation in C. elegans requires the spindle checkpoint.</a> Science. 302, 1038-1041 (2003).</li><li>Margalit, A., Vlcek, S., Gruenbaum, Y., Foisner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+and+making+of+the+nuclear+envelope.">Breaking and making of the nuclear envelope.</a> J. Cell Biochem. 95, 454-465 (2005).</li><li>Maddox, A. S., Maddox, P. 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特別な利害関係は宣言されません。

酸素欠乏と仮死状態深刻な酸素欠乏への曝露は、いくつかの生物にとって致命的なことができますが、いくつかの生物が無酸素への暴露を生き抜いてきた。 Cの場合にはelegansは 、無酸素暴露の生存率は、発達段階や酸素欠乏に対する応答に依存するエントリが観察可能な生物学的プロセスの数が逮捕された仮死状態に可逆的である。酸素は環境7,9に再...

<table border="1"><tbody><tr><td> 試薬/機器 </td><td></td><td></td><td> 構図 </td></tr><tr><td>次亜塩素酸塩溶液</td><td></td><td></td><td> 0.7グラムのKOH、12ミリリットルの5％次亜塩素酸ナトリウムは、蒸留H 2 0で50mlにもたらす</td></tr><tr><td> M9バッファー</td><td></td><td></td><td> 3グラムのKH 2 PO 4、11グラムのNa 2 HPO 4、5gのNaCl、1リットルの蒸留H 2 Oあたり1ミリリットル（1 M）のMgSO 4を </td></tr><tr><td>ガラス顕微鏡スライド</td><td>フィッシャー·サイエンティフィック</td><td> 12-550-343 </td><td> 3 &quot;×1&quot; X1.0さmm </td></tr><tr><td>ラウンドマイクロカバーガラス</td><td>電子顕微鏡学</td><td> 72223から01 </td><td>直径25mm </td></tr><tr><td>ハロカーボン油700 </td><td>シグマ</td><td> H8898-100ML </td><td></td></tr><tr><td>麻酔薬</td><td></td><td></td><td> TRIC 0.5パーセントアイネ、0.05％tetramisole </td></tr><tr><td>ライデンクローズド灌流Microincubator </td><td>ハーバード装置</td><td> 650041 </td><td></td></tr><tr><td>超高純度窒素</td><td> Calgaz（エア·リキード） </td><td></td><td> &gt; 99.9990パーセントのN 2 &lt;2 ppmのO 2 </td></tr><tr><td>プラスチックチューブ</td><td> VWR </td><td> 89068-468 </td><td> 0.062 &quot;内径x 0.125&quot; OD </td></tr><tr><td>ディスク共焦点顕微鏡をスピニング</td><td>マクベインシステムズ</td><td></td><td>ツァイスは光学顕微鏡、落射照明系、CSU-10横河電機共焦点スキャナ、浜松電子増倍CCDカメラを反転します。 </td></tr></tbody></table>

use of time lapse microscopy to visualize anoxia-induced suspended animation in c. elegans embryos

Caenorhabditsエレガンスは、成人と胎児の段階の透明度だけでなく、融合タンパク質1-4の無数を表現する遺伝子変異体およびトランスジェニック系統の可用性によって促進されるストレス耐性、の研究に広く用いられている。また、このような細胞分裂のような動的なプロセスは、蛍光標識したレポータータンパク質を使用して表示できます。有糸分裂の研究は、無傷の生物を含む様々なシステムでタイムラプス実験の使用によって容易にすることができ、したがって初期のC.線虫の胚はよくこの研究に適しています。ここで紹介する無酸素に応答して、それによって細胞内構造のin vivoイメージング技術（99.999％N 2、&lt;2 ppmのO 2）があるストレスが高倍率の顕微鏡でのセットアップを使用した単純なガスの流れを使用することで可能です。 microincubation室が通じ窒素ガスの流れと回転するディスク共焦点顕微鏡と組み合わせて使用​​され動物は、in vivoで画像化することができる管理された環境を作成することができます。 GFPタグガンマチューブリンやヒストン用いて、ダイナミクスと細胞分裂の逮捕は、酸素欠乏環境にさらされている間と後に、前に、監視することができます。この手法の結果は、酸素欠乏にさらされた胚の割球内の高解像度、詳細なビデオや細胞構造のイメージです。

深刻な酸素欠乏（無酸素）にさらさC. elegansの胚は、開発や細胞分裂7を含む生物学的プロセスを停止させることで生き残ることができます。細胞分裂の酸素欠乏によって誘導された逮捕は、動物は、in vivoで画像化することができる管理された環境を作成することによる窒素ガスの流れと回転するディスク共焦点顕微鏡と組み合わせてmicroincubation室を使用することによっ?...

Watch this Scientific Journal Video about Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos at JoVE.com

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

ここで説明<em生体内で&gt;</em回転するディスク共焦点顕微鏡と組み合わせてmicroincubation室を介してガス流を用いた無酸素に暴露された動物の画像サブ細胞構造へ&gt;テクニック。この方法は簡単で、実験パラメータとモデルの様々なシステムに適合するように十分な柔軟性を備えています。

における酸素欠乏によって誘導された仮死状態を可視化するために時間の経過顕微​​鏡の使用<em&gt; Cエレガンス</em&gt;胚

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

use-time-lapse-microscopy-to-visualize-anoxia-induced-suspended

Research

JoVE Journal

Biology

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

11.5K ビュー.  University of North Texas. ここで説明テクニック。この方法は簡単で、実験パラメータとモデルの様々なシステムに適合するように十分な柔軟性を備えています。

ビデオ: Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Monitoring Actin Disassembly with Time-lapse Microscopy

タイムラプス顕微鏡で分解アクチンの監視

生物学

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

ライブゼブラフィッシュの胚における二光子軸索切断とタイムラプス共焦点イメージング

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

siRNAトランスフェクション後の有糸分裂のタイムラプスイメージング

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

イメージング C.エレガンス</em落射蛍光顕微鏡とオープンソースソフトウェアを使用して>胚

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

で遺伝子発現プロファイルを決定 C.エレガンスマイクロアレイとリアルタイムPCRを用いて

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

の初期発生のタイムラプス顕微鏡線虫（Caenorhabditis elegans）

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

の後胚表現型を識別するためのRNAiスクリーニング C.エレガンス</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

タイムラプスビデオ顕微鏡を用いた細胞のランダムな移行の定量分析

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

SOX10中の顔面の開発の可視化：楓トランスジェニックゼブラフィッシュラインタイムラプス共焦点顕微鏡を用いて

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

筋肉の細胞内コンパートメントを評価するための方法<em&gt; C。エレガンス</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

における酸素欠乏によって誘導された仮死状態を可視化するために時間の経過顕微​​鏡の使用

における酸素欠乏によって誘導された仮死状態を可視化するために時間の経過顕微鏡の使用