我々は教授ジュディスアーミテージ（オックスフォード大学、英国）と教授コンラッドMullineaux（ロンドンのクイーンメアリー大学、英国）のグループから、菌株の種類の寄付を認める。 IMDは、共同で生化学の部門（オックスフォード大学）とOCISBによって資金を供給され、ARは、工学物理科学研究評議会（EPSRC）DTCの学生の身分によって資金を供給され、NDは、バイオテクノロジーおよび生物科学研究会議（BBSRC）から資金提供され、MCLは王立協会大学研究フェローシップによって資金を供給。

<ol><li>この手順を開始するには、 大腸菌の細菌細胞を発現する蛍光蛋白質の凍結ストックの50μlを最初に解凍し、午前中は37℃で一晩5 mlのLB培養培地で振盪しながら好気的に栽培されている、この飽和文化の50μlを抽出し、 、最小のM63グルコースの培地へと継代培養4〜6時間、30℃でインキュベートする。ここでは、電子輸送を表現するそのうちの一つ、2つの異なる細胞株を、使用例を示すGFP、GFPと融合した細菌べん毛モーターに関与するタンパク質を発現する他に融合されたチトクローム。 </li><li>細胞は固定化のサンプルとしてそれらを表示する場合のいずれかに成長のサブカルチャーから直接採取したり、それらは&quot;テザー&quot;の条件で表示する場合細菌の鞭毛を切り詰めるために剪断することができます。 </li><li>せん断は、無菌配管で結合された2つの滅菌注射器で構成されるデバイスに典型的にはサブカルチャーの1〜5ミリリットルを置くことが含まれます。せん断は、狭いチューブを通して文化をプッシュするごとにシリンジポンプを押し込んで交互に行われます。これは、必要なせん断の程度に応じ、50〜100倍が行われる。文化は、鞭毛のフラグメントを削除するには、最少培地で再懸濁している細胞をペレットに遠心分離されています。 </li><li>私たちは、その後、その後、脱イオン水とエタノールで徹底的に洗浄し、少なくとも1時間空気中で乾燥するまま、20分間エタノールにKOHの飽和溶液に浸漬することにより、BK7ガラス製カバースリップの洗浄準備</li><li>我々は、顕微鏡で細胞を収容するための簡単​​なフローセルを構築する。これは、BK7ガラスの顕微鏡スライド上にパラフィングリースの描画線を含み、その上にきれいにcoverlipsのいずれかを配置し、ピンセットで軽く押し下げて、トンネルサンドイッチを作成、5〜10μlののフローセルのボリュームを与える。 </li><li>固定化細胞を観察するために我々は、ポリ- L -リジンの0.1％w / v溶液を注射することにより、フローセルを入力し、それは少なくとも1分間室温でインキュベートすることができます。我々は、その後、フローセルの一方の端からメディアを注入し、他からティッシュペーパーでウィッキングで100μlの最少培地を用いて洗い流す。 </li><li>私たちは、その後、カバーガラスの表面をマークする最少培地で200nmの直径ラテックス微小球（Polysciences）の1:500希釈の20μlを通過ウィク。フローセルは、カバースリップは、下向きにシンプルな湿度チャンバーで表面の明確なプラットフォーム上に配置し、5分間室温でインキュベートされるように反転されます。非結合ビーズを100μlの最小限のメディアを介して吸い上げによって洗い流されている。 </li><li>我々がつながれた細胞を観察したい場合は我々は、ポリ- L -リジンのステップを省略し、代わりに5 mg / mlの抗フラジェリン抗体とフローセルを埋める。フローセルは、10分間加湿チャンバー内の場所ですし、発散性に優れたによってによってフラッシュされます。 </li><li>細胞培養液20μlの固定化細胞を見に係留細胞または断片化していない試料を観察する場合のどちらか剪断されたサンプルを使用して、フローセル通過して邪悪です。 </li><li>フローセルが反転し、20分間加湿チャンバーに配置されます。非結合細胞を次いで100μlの最小限のメディアを通して吸い上げによって洗い流されている。 </li><li>液浸オイルのドロップはカバースリップの上面の中央に配置され、フローセルは、高開口数対物レンズとの光学的接触をする、カスタムビルドの蛍光顕微鏡の試料ホルダーにに軽く置かれます。 </li><li>顕微鏡電子乗算カメラがオンになり、カメラは-70℃に冷却するように設定され、ソフトウェアは、最初のゲインコントロールをdisenabling、フレーム転送モードで25Hzの典型的なフレームレートで画像を取得するために設定されていますカメラ。 </li><li>明視野照明のスイッチがオンになっていると画像がそのカバーの表面に平行に、その長軸と立ち往生しているに基づいて撮像するセルの適切なセルまたはグループを選択し、フォーカスに取り込まれます。フォーカスが細かくカバーの表面に付着した200nmのラテックスビーズをジャストフォーカスになっていることを保証するために調整されます。 </li><li>イメージシーケンスは、細胞体の輪郭を記録するために明に取得されます。明視野照明はオフになり、カメラのゲインが最大に有効になっています。 </li><li>内部全反射蛍光（または反射）、適切な波長以上のレーザービームを（ここで、緑色蛍光タンパク質の励起のために、我々は473 nmのレーザーを使用）を使用して標準的な取得のための後焦点面に集束されるようにあらかじめ設定されています対物レンズが、細胞のサンプルに蛍光励起のためのエバネッセント場を生成する光軸から横方向にずれる。 </li><li>カメラの取得が開始され、レーザーのシャッターには、細菌内で蛍光タンパク質を励起するために開かれます。買収のレーザーの強さと速度のパラメータは異なる値を試すことで調査中の特定の生物学的システム向けに最適化する必要がありますが、典型的な範囲R細胞膜におけるモバイルタンパク質複合体を研究するelevantは50から40 msのフレームあたりの露光時間で、直径〜30μm程度の円形の励起領域の上に1から10 mWのレーザパワーです。サンプルは通常、〜10秒間、photobleachedまで点灯されています</li><li>このプロトコルは、両方のセルの本体は、カバースリップと鞭毛スタブとの間の、そして堅くカバーの表面に固定されている固定化細胞のための結合点を中心に回転されるテザーセルに使用することができます。 </li><li>複合体の高コピー数を持ついくつかの細胞株は、前の全反射イメージングにセルの一方の極の初期回折限界の集光漂白剤の恩恵を受ける。この漂白剤は、（またはFRAP）光退色後蛍光回復で使用されているものと同等です。私たちのカスタム顕微鏡を使用して、励起レーザ光の一部は、FRAP型漂白に使用する第2の独立したパスにオフ供給することができます。通常は1 - 10mWのレーザパワーは、範囲10から300ミリ秒の典型的な漂白時間は、使用されています。その後、より簡単に可視化するためにその領域に拡散させる個々の複合体を可能にする細胞の漂白ゾーンではるかに高いイメージングコントラストでこの結果。 </li><li> &quot;slimfield&quot;と呼ばれる別の照明モードが採用されている細胞膜に比べてはるかに高速拡散細胞の細胞質で複合体を視覚化するための。ここで焦点レーザーは、横方向にちょうど単一のセルを含むように拡張されている。これは通常、ミリ秒のかなり高速のエクスポージャーを取るこ​​とができ、非常に強烈なフィールドを生成します。 </li><li>データ取得に続いて、画像はカスタム作成されたソフトウェア（LabVIEW 8.5でコード化された）に供給される。これは自動的に通常、数ナノメートルの精度に細胞内の蛍光スポットの位置を検出し、その大きさと明るさを抽出します。追跡分子複合体の時間に対する光退色トレースはその後化学量論を推定するために使用されているbrightnessofは、単一の分子複合体を構成する方法多くの個々の蛍光タンパク質、すなわち。 </li></ol>代表的な結果： プロトコルが正しく行われている場合、明視野で見る細胞の画像は白/グレーの細胞体（図1a）に対して暗細胞体の周囲で、非常に区別される。蛍光を使用して固定化細胞では、我々は、幅で、通常は250から300 nmの（図1b）の別個のスポットの強度を、見ることができます。健康、テザ​​ー細胞は、明視野像におけるテザーの添付ファイルのポイントを中心に回転するように見られます。蛍光励起の下で我々の場合におけるいくつかの分子複合体はまた、べん毛モーターを持つタグタンパク質の局在を示す、添付ファイルの時点で見られるかもしれません。これらのスポットは、個々の分子の複合体であると見られるそれらの数は、使用される照明モードに依存し、実際に一度に細胞内でどのように多くの複合体の提示されています。スポットの移動度は、研究対象の特定の生物学的システムに依存します。スポットの密度が最初は非常に高い場合は、ここで使用ラベル付けシトクロムの場合と同様に、初期FRAPの漂白を行うと、画像のコントラストを向上させることができます。 <img src="/files/ftp_upload/1508/1508Figure1tmb.jpg" border="0" alt="図1" /> 図1。細菌の鞭毛モーターに関与することが知られている緑色蛍光タンパク質（GFP）に融合タンパク質を発現する固定化大腸菌の細胞のための（A）明視野と（B）全反射の画像（疑似カラー）。してください<a href="https://www.jove.com/files/ftp_upload/1508/1508Figure1.jpg">ここをクリックして</a>図1の拡大バージョンを参照すること。

<ol>
	<li>Leake, M. C., Chandler, J. H., Wadhams, G. H., Bai, F., Berry, R. M., Armitage, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometry+and+turnover+in+single,+functioning+membrane+protein+complexes.">Stoichiometry and turnover in single, functioning membrane protein complexes.</a> Nature. 443, 355-358 (2006).</li><li>Leake, M. C., Greene, N. P., Godun, R. M., Granjon, T., Buchanan, G., Chen, S., Berry, R. M., Palmer, T., Berks, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable+stoichiometry+of+the+TatA+component+of+the+twin-arginine+protein+transport+system+observed+by+in+vivo+single-molecule+imaging.">Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging.</a> Proc Natl Acad Sci U S A. 105, 15376-15381 (2008).</li><li>Leake, M. C., Wilson, D., Gautel, M., Simmons, R. M., M, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elasticity+of+single+titin+molecules+using+a+two-bead+optical+tweezers+assay.">The elasticity of single titin molecules using a two-bead optical tweezers assay.</a> Biophys. J. 87, 1112-1135 (2004).</li><li>Leake, M. C., Wilson, D., Bullard, B., Simmons, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elasticity+of+single+kettin+molecules+using+a+two-bead+laser-tweezers+assay.">The elasticity of single kettin molecules using a two-bead laser-tweezers assay.</a> FEBS Lett. , 535-555 (2003).</li><li>Lenn, T., Leake, M. C., Mullineaux, C. W. <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+clustering+and+dynamics+of+cytochrome+bd+complexes+in+the+Escherichia+coli+plasma+membrane.">In vivo clustering and dynamics of cytochrome bd complexes in the Escherichia coli plasma membrane.</a> Mol. Microbiol. 70, 1397-1407 (2008).</li><li>Lenn, T., Leake, M. C., Mullineaux, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+Escherichia+coli+OXPHOS+complexes+concentrated+in+specialised+zones+within+the+plasma+membrane.">Are Escherichia coli OXPHOS complexes concentrated in specialised zones within the plasma membrane.</a> Biochem. Soc. Trans. 36, 1032-1036 (2008).</li><li>Lo, C. J., Leake, M. C., Pilizota, T., Berry, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+measurements+of+Membrane+Potential,+Sodium-Motive+Force+and+Flagellar+Motor+Speed+in+Escherichia+coli.">Single-cell measurements of Membrane Potential, Sodium-Motive Force and Flagellar Motor Speed in Escherichia coli.</a> Biophys. 93, 294-302 (2007).</li><li>Lo, C. J., Leake, M. C., Berry, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+measurement+of+intracellular+sodium+concentration+in+single+Escherichia+coli+cells.">Fluorescence measurement of intracellular sodium concentration in single Escherichia coli cells.</a> Biophys. J. 90, 357-3565 (2006).</li></ol>

The authors have nothing to disclose.

これは、べん毛モーターの機能を損なうおそれがあるので注意が、つながれた細菌を調べるための細胞&quot;せん断を介して&quot;しないように注意する必要があります。彼らは酸素枯渇になる可能性があるので、顕微鏡スライド上に、1時間に1回よりもはるかに長いためにセルを使用することが重要です。かなりの最適化は、最適な顕微鏡の撮像条件が検討され、特定の生物学的システムへの?...

visualizing single molecular complexes in vivo using advanced fluorescence microscopy

生きた細胞のメカニズムへの完全な洞察は、細胞レベルでの事象を惹起し、ダイレクトキープロセスを調べることによってのみ達成することができます。生物系のせん断複雑さをこれまでには、代わりに相対的に原油バルクアンサンブル平均測定を使用して単一システムの研究に焦点を当て、あまりにも要求される正確な単一分子実験を引き起こしている。しかし、多くの重要なプロセスは一つのレベルで生きている細胞や少数の分子で起こる、アンサンブルの測定値は、一般的にこれらのイベントの確率と異種の性質を覆い隠す。ここでは、高度な光学顕微鏡と我々は、単一の分子とどのように我々は生物学的機械の機能に分子複合体内動態を観察できるの精度への単一の生きた細菌細胞内のタンパク質を監視する方法を示す分析画像解析ツールを使用して。テクニックは、生理学的に直接関連しています。彼らが検討されて生体試料に最小限の摂動と非侵襲的であり、完全に生体を構成する物質の調査、生物物理学の他の単一分子のアプローチが容易に利用できない機能に同調されています。さらに、生物学的標本は、すべて自然に発生するよりもはるかに多くのタンパク質を生成するための、より一般的ですが、理想的なアプローチとは対照的に変更されていない細胞株（&quot;ゲノムエンコード&quot;）とほとんど同一のレベルで、少なくとも蛍光タグタンパク質を生成する研究（&quot;発現プラスミド&quot;）。このように、調査される実際の生物学的サンプルは、自然の生物にかなり近いですので、実際の生理学的プロセスへの観察は、より関連性の高い。

Watch this Scientific Journal Video about Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy at JoVE.com

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

ここでは、機能性分子複合体を追跡し、定量、検出できるようにするために細菌細胞を生きて単一分子蛍光顕微鏡を実行するためのプロトコルを示す。

単一分子複合体の可視化インビボでの高度な蛍光顕微鏡を用いて

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

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Research

JoVE Journal

Biology

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

9.2K ビュー.  University of Oxford. ここでは、機能性分子複合体を追跡し、定量、検出できるようにするために細菌細胞を生きて単一分子蛍光顕微鏡を実行するためのプロトコルを示す。

ビデオ: Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

蛍光顕微鏡による可視化単分子のDNAの複製

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

生物学

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 ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

蛍光顕微鏡を用いて線虫の中の細菌の可視化

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

高度な3D蛍光顕微鏡を用いたオートファジーの定量分析

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a &#34;double decker&#34; microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm.

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

The giant ciliate Stentor coeruleus is a classical system for studying regeneration and wound healing in single cells.&#160;By imaging Stentor cells simultaneously at low and high magnification it is possible to measure cytoplasmic flows before, during, and after wounding.

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

迅速な分析および蛍光顕微鏡画像の探索

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

蛍光顕微鏡でストロミュール頻度を可視化

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

3 D 蛍光顕微鏡を用いたウイルス転写工場の機能イメージング

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

生体共焦点顕微鏡を用いてリンパ節構造と細胞局在化を可視化

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

蛍光生細胞顕微鏡によるミトティック・マイオシス酵母核ダイナミクスの検討

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...