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>

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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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

11.6K 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

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

のためのシンプルなマイクロ流体デバイス<em生体内で&gt;</emの&gt;イメージング<em&gt; Cエレガンス</em&gt;<em&gt;ショウジョウバエ</em&gt;とゼブラフィッシュ

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

生物工学

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

等方性光シート顕微鏡と自動細胞系譜分析による、細胞内分解能を Caenorhabditis elegans 胚発生

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

発生生物学

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development&#160;and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.

中に神経芽細胞細胞質分裂を可視化する<em&gt; C.虫</em&gt;胚

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...

神経科学

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

C.エレガンス胚発生を解析するセミハイスループットイメージング法とデータ可視化ツールキット

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans has emerged as one of the best models for studying development. From an optical point of view, its size and transparent body make this nematode an ideal specimen for DIC (Differential Interference Contrast or Nomarski) microscopy. This article illustrates a protocol for growing C. elegans nematodes, preparing and mounting their embryos, performing 4D microscopy and cell lineage tracing. The method is based on multifocal time-lapse records of Nomarski images and analysis with specific software. This technique reveals embryonic developmental dynamics at the cellular level. Any embryonic defect in mutants, such as problems in spindle orientation, cell migration, apoptosis or cell fate specification, can be efficiently detected and scored. Virtually every single cell of the embryo can be followed up to the moment the embryo begins to move. Tracing the complete cell lineage of a C. elegans embryo by 4D DIC microscopy is laborious, but the use of specific software greatly facilitates this task. In addition, this technique is easy to implement in the lab. 4D microscopy is a versatile tool and opens the possibility of performing an unparalleled analysis of embryonic development.

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

Here, we present a protocol for preparing and mounting Caenorhabditis elegans embryos, recording development under a 4D microscope and tracing cell lineage.

4D顕微鏡:ノマルスキ顕微鏡を用いた カエノラブディティス・エレガンス 胚発生の解明

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans ...

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

新規メトリックは、線虫の胚伸びを特徴づけるために、<em&gt;線虫（Caenorhabditis elegans）</em

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

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 ...

生物学

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 ...

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

カエノラブディティス・エレガンスの長期増殖とイメージングのためのシンプルなマイクロ流体チップ

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

における虚血/再灌流のための無酸素飢餓モデル<em&gt; C.虫</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

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

要約

さらに動画を探す

この動画の章

のためのシンプルなマイクロ流体デバイス

等方性光シート顕微鏡と自動細胞系譜分析による、細胞内分解能を Caenorhabditis elegans 胚発生

中に神経芽細胞細胞質分裂を可視化する

C.エレガンス胚発生を解析するセミハイスループットイメージング法とデータ可視化ツールキット

4D顕微鏡:ノマルスキ顕微鏡を用いたカエノラブディティス・エレガンス胚発生の解明

新規メトリックは、線虫の胚伸びを特徴づけるために、

イメージング C.エレガンス胚

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

カエノラブディティス・エレガンスの長期増殖とイメージングのためのシンプルなマイクロ流体チップ

における虚血/再灌流のための無酸素飢餓モデル

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

要約

さらに動画を探す

この動画の章

のためのシンプルなマイクロ流体デバイス

等方性光シート顕微鏡と自動細胞系譜分析による、細胞内分解能を Caenorhabditis elegans 胚発生

中に神経芽細胞細胞質分裂を可視化する

C.エレガンス胚発生を解析するセミハイスループットイメージング法とデータ可視化ツールキット

4D顕微鏡:ノマルスキ顕微鏡を用いた カエノラブディティス・エレガンス 胚発生の解明

新規メトリックは、線虫の胚伸びを特徴づけるために、

イメージング C.エレガンス胚

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

カエノラブディティス・エレガンスの長期増殖とイメージングのためのシンプルなマイクロ流体チップ

における虚血/再灌流のための無酸素飢餓モデル

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

4D顕微鏡:ノマルスキ顕微鏡を用いたカエノラブディティス・エレガンス胚発生の解明