我々は、技術的なアドバイスのために生物物理化学、ドイツとDavid J.サンドストローム、国立精神衛生研究所、国立衛生研究所、ベセスダ、MD、米国のためにアンドレアスSchönle、マックスプランク研究所に感謝する。我々は、イメージング室と麻酔のデバイスを構築するためのフランクKötting、ヨーロッパの神経科学研究所、ゲッティンゲンに感謝。この作品は、アルツハイマー病ForschungイニシアティブからTMRYZへの補助金によって支えられている細胞分子神経科学、テュービンゲン大学の大学院の交わりによってSBH、中国奨学金委員会の交わりによってサポートされていました。

イメージングチャンバーのA）組立<ol><li>選択された段階（25℃で約24時間の観測間隔をあけ° Cの段階を徘徊するまで、例えば初期の第3齢幼虫）の幼虫を選択します。 </li><li>培養液し、乾燥DABのそれをきれいに水で幼虫をすすぎます。 </li><li>コー​​トチャンバの底部の要素の中心、ハロカーボンオイルの薄膜で、幼虫に直面する地域。 </li><li>チャンバー内の顕微鏡対物（神経筋接合部（NMJ）26および27は、イメージを作成することができます）（図1 AE）に面した腹側に幼虫を置く。 </li><li>グリッド面の上方（図1）の空気のスロットで、油の層の上にプラスチック製のスペーサを置きます。このステップで確認してください：スペーサの高さは約半分の幼虫の直径であり、スリットの幅は、幼虫の約2倍の直径にする必要があります。 </li><li>顕微鏡でイメージング室を配置</li></ol>幼虫のB）麻酔<ol start="7"><li>適切な麻酔装置/気化器とチャンバーの二つのインレットを接続します。 アップフロントを確認してください。 室内空気中のデスフルランの有効濃度は、安全規則によって指定されたものを超えないようにしてください！デスフルランのごくわずかな総容量は、幼虫を麻酔するために必要です。デスフルラン1の15％のアプリケーション（v / v）の実験で使用する理想的な濃度を決定するための良い出発点であることが証明されている。安全上の理由から、我々は、気化器は、in vivoイメージングの2-4時間のために必要以上のデスフルランが含まれてはならないことを示唆している。 </li><li>水のガラスでチャンバーの出口に接続されたチューブのもう一方の端を浸してから約5秒間チャンバーへの麻酔の流れを制御するバルブを開きます。 このステップで確認してください。 気泡が水に登るのですか？そうでない場合チャンバーは漏れやすいです。 </li><li>約3秒間のバルブを閉じます。 </li><li>顕微鏡で残留幼虫の動きを監視します。ハートビートと筋肉の動きの両方を確認してください。幼虫の麻酔が完了するまでに必要なオープンとクローズのバルブは、ステップ8および9で説明されている場合は、すべてのバルブを閉じて、イメージングを起動します。 このステップで確認してください。 残留筋肉の動きやハートビートは、幼虫が適切に麻酔ではないことを示します。完全な麻酔は、高解像度の画像が不可欠です。幼虫は通常、特定の時点よりも長い15〜20分のための麻酔であってはならない。 </li></ol> C）イメージング<ol start="11"><li>正しい位置と画像の興味の構造（図2-4）を識別する。 </li></ol> D）麻酔からの回復は<ol start="12"><li>イメージングは​​、チャンバー内に空気を聞かせ完了後。 このステップで確認してください。 送信されるハロゲンの光によって幼虫のハートビートと筋肉の収縮を確認してください。筋肉が収縮を始めると、それは、イメージング室から幼虫を削除しても安全です。 </li><li>麻酔装置からチャンバーを外し、顕微鏡から取り外します。慎重にチャンバーを分解しますとマッシュポテトフライ培養培地に幼虫を置く。 </li><li>適切な温度でインキュベーター内で料理を保管してください。 </li></ol> E）時系列<ol start="15"><li>十分な時間のポイントが得られるまで手順2-14を繰り返します。 代替プロトコル： 幼虫は30分間隔内で複数回撮像する場合には、次の麻酔の時点まで、イメージングチャンバー内にそれを残すために実用的かもしれません。十分な時間のポイントが得られるまで手順7-14を繰り返します。 このステップで確認してください。 幼虫は一度に2時間以上の撮像室内に保持されるものではない、またその時点でより長い15〜20分のための麻酔になることです。 </li></ol><img src="/files/ftp_upload/2249/2249fig1.jpg" alt="図1" /> イメージングチャンバーの1アセンブリを示します 。 （A）幼虫と油の層の上にプラスチック製のスペーサを置きます。 （B）、次のスペーサーで22 × 22 mmのカバースリップを置き、チャンバー内にプレキシグラスのガイドリングを挿入する（C）金属リングと幼虫の位置を修正し、（D）チャンバーを閉じます。 （E）今すぐチャンバーは顕微鏡にマウントする準備が整いました。 <img src="/files/ftp_upload/2249/2249fig2.jpg" alt="図2" /> ショウジョウバエの幼虫の2ボディー壁の筋肉を図 。筋肉やNMJsはCD8 - GFP - SH融合蛋白質8の発現によって可視化した。幼虫にクチクラを通して腹側に焦点を当てたときに観察されるように筋肉が表示されます。 （AC）で最も表面的な筋肉、27は、最も内側の筋肉、6と7は、表示されている（KL）で、示されています。スケールバー：100μmの、スライス間のΔZは2μmである。 <img src="/files/ftp_upload/2249/2249fig3.jpg" alt="図3" /> ショウジョウバエの幼虫の体壁筋の3アイデンティティを示しています 。 L3 ショウジョウバエの幼虫の筋肉のアイデンティティ、セグメントのA3は、示されている。筋肉やNMJsはCD8 - GFP - SH融合蛋白質8の発現によって可視化した。幼虫にクチクラを通して腹側に焦点を当てたときに観察されるように筋肉が表示されます。 ACで最も表面的な筋肉、27は、表示され、KLで最も内側の筋肉、6と7で、示されています。スケールバー：100μm以下、スライス間のΔZは2μmである。 <img src="/files/ftp_upload/2249/2249fig4.jpg" alt="図4" /> 図ショウジョウバエの幼虫の神経筋接合部の4アイデンティティ 。 （AD）NMJsはDGluRIIA - MRFP融合タンパク質1の発現によって可視化した。幼虫にクチクラを通して腹側に焦点を当てたときに観察されるようにNMJsが表示されます。 （AB）の最も浅NMJ、27日に示されている、CDの中で最もインテリアNMJs、6と7は、表示されます。スケールバー：100μm以下、スライス間のΔZは5μmである。 （E）と（F）表層（E）とより多くのインテリア（F）NMJsのアイデンティティは参照のために与えられます。

<ol>
	<li>Rasse, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+receptor+dynamics+organizing+synapse+formation+in+vivo.">Glutamate receptor dynamics organizing synapse formation in vivo.</a> Nat Neurosci. 8, 898-905 (2005).</li><li>Rasse, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In vivo imaging of long-term changes in the Drosophila neuromuscular system [dissertation]. , (1988).</li><li>Fuger, P., Behrends, L. B., Mertel, S., Sigrist, S. J., Rasse, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+synapse+development+and+measuring+protein+dynamics+using+two-color+fluorescence+recovery+after+photo-bleaching+at+Drosophila+synapses.">Live imaging of synapse development and measuring protein dynamics using two-color fluorescence recovery after photo-bleaching at Drosophila synapses.</a> Nat Protoc. 2, 3285-3298 (2007).</li><li>Wang, X., Schwarz, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+axonal+transport+of+mitochondria.">Imaging axonal transport of mitochondria.</a> Methods in enzymology. 457, 319-333 (2009).</li><li>Vinegoni, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesoscopic+fluorescence+tomography+for+in-vivo+imaging+of+developing+Drosophila.">Mesoscopic fluorescence tomography for in-vivo imaging of developing Drosophila.</a> J Vis Exp. , (2009).</li><li>Lin, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+imaging+of+Drosophila+larva+by+multiphoton+autofluorescence+and+second+harmonic+generation+microscopy.">Label-free imaging of Drosophila larva by multiphoton autofluorescence and second harmonic generation microscopy.</a> Journal of biomedical optics. 13, 050502-050502 (2008).</li><li>Levitan, E. S., Lanni, F., Shakiryanova, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+vesicle+motion+and+release+at+the+Drosophila+neuromuscular+junction.">In vivo imaging of vesicle motion and release at the Drosophila neuromuscular junction.</a> Nat Protoc. 2, 1117-1125 (2007).</li><li>Zito, K., Parnas, D., Fetter, R. D., Isacoff, E. Y. &. a. m. p. ;. a. m. p., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Watching+a+synapse+grow:+noninvasive+confocal+imaging+of+synaptic+growth+in+Drosophila.">Watching a synapse grow: noninvasive confocal imaging of synaptic growth in Drosophila.</a> Neuron. 22, 719-729 (1999).</li></ol>

利害の衝突は宣言されません。

提示方法は、最初にキイロショウジョウバエの幼虫の体壁筋上のグルタミン酸作動性シナプスを研究するために開発されました。 ショウジョウバエ神経筋接合部（NMJ）は、筋肉や神経細胞のステレオタイプCYTO -アーキテクチャによって特徴付けられるため、理想的にin vivoイメージングに適しています。しかし、説明した麻酔のプロトコルは、イメージングNMJに限定されるものではなく、 ショウジョウバエの幼虫の透明性は、器官の発達を研究するために記述されたプロトコルの適応、細胞の遊走、細胞内の軸索の貨物とサブセルラー再編の輸送を容易にします。

試薬： <ul><li>デスフルラン（Suprane、バクスター、ウンターシュライスハイム、ドイツ） </li><li>ハロカーボンのオイル（例えばVoltalef H10S油/アトフィナ、Puteaux、フランス） </li><li>培地を飛ぶ</li></ul>設備： <ul><li>倒立型共焦点顕微鏡</li><li>双眼顕微鏡（例えばSTEMI 2000、カールツァイス、イエナ、ドイツ） </li><li>並べ替えに使用される小さな絵筆が飛ぶ</li><li>インキュベーター</li><li>カスタム構築されたイメージング室（機械図面のrefを参照してください。3） </li><li>気化器/麻酔装置（詳細は文献3参照） </li></ul>

in vivo imaging of intact drosophila larvae at sub-cellular resolution

光学イメージングの最近の改善は、希望するトランスジェニック動物のラインの効率的な設置を可能にする遺伝的に符号化された蛍光体と遺伝学的ツールは、生活の文脈で検討する生物学的プロセスを有効にしている、といくつかのインスタンスでも、生物を行動する。このプロトコルでは我々は1-3のサブ携帯の解像度でシナプス集団の発達と可塑性に従うことをデスフルラン揮発性麻酔薬を、使用して、無傷のショウジョウバエの幼虫を麻酔する方法について説明します。 キイロショウジョウバエの幼虫を麻酔するために他の有用なメソッドが以前に4,5,6,7,8を説明してきたが、本明細書に提示プロトコルは、以下の複合キーの機能に起因する大幅な改善を示しています：麻酔の（1）非常に高い学位を、さらにハートビートが2日目および（する時間の期間にわたって個以上の時間ポイントの記録を可能にして、麻酔のサイクルごとに&gt; 90％の（2）高い生存率、最大150 nmの1の横方向の解像度を可能に逮捕される3）2つのインスタンスで私たちを可能にする高感度は、生理学的レベルでの発現タンパク質のダイナミクスを研究する。具体的には、我々は、GluR - IIAは安定したトランスジェニック系統における内因性プロモーター1とエキソントラップラインFasII - GFP 1を介して発現するシナプスのグルタミン酸受容体サブユニットを可視化することができた。 （4）他の方法とは対照的に4,7幼虫がないだけ生きてイメージを作成するだけでなく、そのまますることができます（すなわち、非解剖）の観察は1日目の数以上発生することができます。付属のビデオの詳細vivoイメージング室2,3 での個々の部品の機能、幼虫、麻酔の手順、方法幼虫内の特定の位置を再認識すると幼虫の安全な取り外しの取付正しい画像からチャンバー。

Watch this Scientific Journal Video about In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution at JoVE.com

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

このプロトコルは、無傷の麻酔とイメージングのための信頼性の高い方法を説明します<em&gt;キイロショウジョウバエ</em&gt;幼虫。我々は数日までのサブセルラー分解能と構造の再認識で反復的なイメージングを可能にするためにデスフルラン揮発性麻酔薬を利用してきた<sup&gt; 1</sup&gt;。

サブセルラー解像度で手つかずのショウジョウバエの幼虫の in vivoイメージングで

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

in-vivo-imaging-of-intact-drosophila-larvae-at-sub-cellular-resolution

Research

JoVE Journal

Biology

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

14.5K ビュー.  University of Tübingen. このプロトコルは、無傷の麻酔とイメージングのための信頼性の高い方法を説明しますキイロショウジョウバエ幼虫。我々は数日までのサブセルラー分解能と構造の再認識で反復的なイメージングを可能にするためにデスフルラン揮発性麻酔薬を利用してきた 1。

ビデオ: In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

ショウジョウバエの幼虫の匂い駆動型の動作の高分解能測定

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

生物学

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

MRイメージングと生体内追跡非侵襲のための酸化鉄ナノ粒子で標識ヒトES細胞とhMSCs

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

アダルトキイロショウジョウバエのロコモーションの高解像度のビデオトラッキング

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

のためのメゾスコピック蛍光トモグラフィー生体内</em開発の>イメージングショウジョウバエ</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

ショウジョウバエの胚の生体内の遠心

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

リビングでのイメージング、細胞の形状の変更ショウジョウバエ胚

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Microgavage of Zebrafish Larvae

The zebrafish has emerged as a powerful model organism for studying intestinal development1-5, physiology6-11, disease12-16, and host-microbe interactions17-25. Experimental approaches for studying intestinal biology often require the in vivo introduction of selected materials into the lumen of the intestine. In the larval zebrafish model, this is typically accomplished by immersing fish in a solution of the selected material, or by injection through the abdominal wall. Using the immersion method, it is difficult to accurately monitor or control the route or timing of material delivery to the intestine. For this reason, immersion exposure can cause unintended toxicity and other effects on extraintestinal tissues, limiting the potential range of material amounts that can be delivered into the intestine. Also, the amount of material ingested during immersion exposure can vary significantly between individual larvae26. Although these problems are not encountered during direct injection through the abdominal wall, proper injection is difficult and causes tissue damage which could influence experimental results. We introduce a method for microgavage of zebrafish larvae. The goal of this method is to provide a safe, effective, and consistent way to deliver material directly to the lumen of the anterior intestine in larval zebrafish with controlled timing. Microgavage utilizes standard embryo microinjection and stereomicroscopy equipment common to most laboratories that perform zebrafish research. Once fish are properly positioned in methylcellulose, gavage can be performed quickly at a rate of approximately 7-10 fish/ min, and post-gavage survival approaches 100% depending on the gavaged material. We also show that microgavage can permit loading of the intestinal lumen with high concentrations of materials that are lethal to fish when exposed by immersion. To demonstrate the utility of this method, we present a fluorescent dextran microgavage assay that can be used to quantify transit from the intestinal lumen to extraintestinal spaces. This test can be used to verify proper execution of the microgavage procedure, and also provides a novel zebrafish assay to examine intestinal epithelial barrier integrity under different experimental conditions (e.g. genetic manipulation, drug treatment, or exposure to environmental factors). Furthermore, we show how gavage can be used to evaluate intestinal motility by gavaging fluorescent microspheres and monitoring their subsequent transit. Microgavage can be applied to deliver diverse materials such as live microorganisms, secreted microbial factors/toxins, pharmacological agents, and physiological probes. With these capabilities, the larval zebrafish microgavage method has the potential to enhance a broad range of research fields using the zebrafish model system.

We present a novel method for microgavage of larval zebrafish utilizing standard embryo microinjection and stereomicroscopy equipment. We demonstrate that microgavage is a safe and efficient technique useful for delivering controlled amounts of diverse materials specifically into the larval zebrafish intestinal lumen.

ゼブラフィッシュ幼生のMicrogavage

The zebrafish has emerged as a powerful model organism for studying intestinal development1-5, physiology6-11, disease12-16, and host-microbe ...

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  ...

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

齧歯気道上皮における繊毛の運動性の高分解能イメージングのためのex vivo法

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

シングルミトコンドリアスーパーオキシド無傷ハートや点滅の共焦点イメージング<em&gt;インビボ</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...