この作品は、健康補助金R01 CA140257-01の国立研究所の国立がん研究所によってサポートされています。

<html> 注：インフォームドコンセントを患者から得られた。また、この研究では、人間の福祉のためのすべて、制度、国、国際的なガイドラインを遵守して行われている。 1。コンピュータの準備<ol><li>上のノートパソコンの電源を入れ、DVI2USBキャプチャカードからUSBを接続してください。 </li></ol> 2。モニターを準備<ol><li>内視鏡医がこれらの画面ではなく、コンピュータからビデオを見ることができるように監視し、PinPのためにDVIケーブルを接続します。 </li><li>スタンディングモニターがついているとDVIに設定されていることを確認します。 </li><li>壁に取り付けられた大型モニターに画像を表示するには、オリンパスのシステムに入力されたボタンを押しオリンパスシステムの背面に子画面を接続します。 </li></ol> 3。レーザダイオードドライバの設定<ol><li>レーザダイオードドライバが動作していないか確認してください。イメージングが実行される前に、それだけで数分をオンにする必要があります。 </li></ol>e_title &quot;&gt; 4。パワーストリップ<ol><li>電源タップにラップトップ、プロセッサ、レーザダイオードドライバ、DVIスプリッタ、およびepiphanキャプチャカードを接続します。 </li></ol> 5。パソコンのデスクトップ上で、MDE広視野を実行します<ol><li> 「現在のフォルダを &#39;ヒット。 </li><li> 「患者のフォルダの作成」の患者数やイニシャル、Enterキーを押します。 </li></ol> 6。キャップを準備し、フィルタ<ol><li>フィルタを取り扱う際は、常にフィルターに油やゴミの移動を最小限に抑えるために手袋、キムワイプを使用しています。ガーゼやアルコール綿棒は、撮影を妨害する可能性の繊維を残すことがあります。 </li><li>フィルタとキャップを下に置くためのプラットフォームを作成するためにテーブルの上にいくつかのキムワイプを配置します。この消毒エリアは、処置中に胃腸科へ簡単にアクセスできる必要があります。 </li><li>フィルタキャップを下に置き、手術中の使用のため近くキムワイプを保つ。 </li></ol> 7。患者の準備<ol><li>使用上の同意の患者遠藤スイートに到着する前に、VFIおよびプロフラビン色素の。 </li><li>上部内視鏡検査の手順のための位置に患者。 </li><li>マルチスペクトルデジタル顕微鏡（MDM）7を使用して、標準的な白色光画像化を進める。 </li></ol> 8。プロフラビンを挿入して、スプレー<ol><li>白色光画像化して終了した後、目的の組織を介してプロフラビン色素を挿入してスプレーします。 1〜5ミリメ​​ートルは、バレット組織の領域に応じて十分なものでなければならない。このステップでプロフラビン染料を噴霧することにより、（少なくとも1分）十分な時間がVFI前に十分な組織吸収のために与えられる。 FDA（IND 102217）からの治験新薬申請の対象とされているプロフラビンは、まもなく適用後の細胞核内に集中する蛍光造影剤である。 VFIは、個々の細胞形態、組織の上のレーザダイオードパンを解決することはできませんが、反射された光は、全体の腺morpholoを鑑賞する内視鏡医が可能になりGY。 </li></ol> 9。レーザダイオードをオンにする<ol><li>レーザダイオードをオンにします。イメージングの開始前に、この少なくとも2分の操作を行います。 </li></ol> 10。VFIのために内視鏡を準備<ol><li>完全に内視鏡を撤回。 </li><li>内視鏡を扱う前には、アシスタントが上の手袋の2ペアを持っていることを確認してください。 </li><li>内視鏡医が上に詳述キムワイププラットフォームの隣にあり、アシスタントの前に内視鏡を保持している。 </li><li>キムワイプで、アシスタントは、内視鏡の先端を清掃する必要があります。優しく前面をきれいにし、また、より簡単に処理するため、内視鏡の側面に沿って数センチをきれいにしてください。 </li><li>洗浄後は、手袋の外側のペアのアシスタント廃棄する場合があります。 </li><li>アシスタントは、フィルターに入れている。内視鏡での補完的な穴にフィルター上の短円筒突起を挿入します。プロセスを支援するために、垂直視鏡を保管してください。 </li><li> FILTを保持するER場所で、フィルターでキャップをスライドさせ、内視鏡の先端にそれを押し下げます。キャップは、安全で、内視鏡と同一平面エッジに完全にプッシュされていることを確認します。キャップのリップがわずかに内視鏡の先端に及ぶことを確認してください。最後に、フィルタは、内視鏡の先端で場所とフラッシュにまだあることを確認してください。 </li></ol> 11内視鏡食道に戻ると画像を挿入 12。食道から内視鏡を削除<ol><li>キムワイプを使用すると、慎重にキャップを引いて、内視鏡フィルタOFF。キャップを捨てて、次のケースのためのフィルターを清掃してください。 </li></ol> 13クリーンフィルター<ol><li>優しくキムワイプでフィルターを清掃してください。 </li><li> Cydexの小さなカップを記入し、12分間のフィルタを沈める。ごとに数分では、フィルターを裏返します。 </li><li>優しくキムワイプでフィルターを清掃してください。 </li><li>滅菌水での今回のフィルタを沈める。 5分後、噴出bを使用しottle軽くフィルター上で滅菌水をスプレーします。 </li><li>少数キムワイプにフィルタを置き、乾燥させます。 </li><li>乾燥したら、キムワイプの間で保存容器に入れます。 </li></ol>

<ol>
	<li>Modiano, N., Gerson, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barrett's+Esophagus:+Incidence,+etiology,+pathophysiology,+prevention+and+treatment.">Barrett's Esophagus: Incidence, etiology, pathophysiology, prevention and treatment.</a> Therapeutics and Clinical Risk Management. 3, 1035-1145 (2007).</li><li>Brown, L. M., Devesa, S. S., Chow, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+Adenocarcinoma+of+the+esophagus+among+white+Americans+by+sex,+stage,+and+age.">Incidence of Adenocarcinoma of the esophagus among white Americans by sex, stage, and age.</a> Journal of the National Cancer Institute. 100, 1184-1187 (2008).</li><li>Siegel, R., Naishadham, D., Ahmedin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Statistics,+2012.">Cancer Statistics, 2012.</a> A Cancer Journal for Clinicians. 62, 10-29 (2012).</li><li>Vieth, M., Ell, C., Gossner, L., May, A., Stolte, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+analysis+of+endoscopic+resection+specimens+from+326+patients+with+Barrett's+esophagus+and+early+neoplasia.">Histological analysis of endoscopic resection specimens from 326 patients with Barrett's esophagus and early neoplasia.</a> Endoscopy. 36, 776-781 (2004).</li><li>Pohl, H., Rosch, T., Vieth, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniprobe+confocal+laser+microscopy+for+the+detection+of+invisible+neoplasia+in+patients+with+Barrett's+oesophagus.">Miniprobe confocal laser microscopy for the detection of invisible neoplasia in patients with Barrett's oesophagus.</a> Gut. 57, 1648-1653 (2008).</li><li>Thekkek, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+video+endoscopy+for+in+vivo+cross-polarized+and+vital-dye+fluorescence+imaging+of+Barrett's-associated+neoplasia.">Modular video endoscopy for in vivo cross-polarized and vital-dye fluorescence imaging of Barrett's-associated neoplasia.</a> Journal of Biomedical Optics. 18, 026007 (2013).</li><li>Roblyer, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multispectral+optical+imaging+device+for+in+vivo+detection+of+oral+neoplasia.">Multispectral optical imaging device for in vivo detection of oral neoplasia.</a> Journal of Biomedical Optics. 13, 024019 (2008).</li><li>Egger, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biopsy+surveillance+is+still+necessary+in+patients+with+Barrett's+oesophagus+despite+new+endoscopic+imaging+techniques.">Biopsy surveillance is still necessary in patients with Barrett's oesophagus despite new endoscopic imaging techniques.</a> Gut. 52, 18-23 (2003).</li><li>Thekkek, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vital-dye+enhanced+fluorescence+imaging+of+GI+mucosa:+metaplasia,+neoplasia,+inflammation.">Vital-dye enhanced fluorescence imaging of GI mucosa: metaplasia, neoplasia, inflammation.</a> Gastrointestinal Endoscopy. 75, 877-887 (2012).</li><li>Muldoon, T. J., Anandasabapathy, S., Maru, D., Richards-Kortum, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+imaging+in+Barrett's+esophagus:+a+novel,+low-cost+endoscopic+microscope.">High-resolution imaging in Barrett's esophagus: a novel, low-cost endoscopic microscope.</a> Gastrointestinal Endoscopy. 68, 737-744 (2008).</li></ol>

開示することは何もありません。

良性上皮化生が高度異形成と腺癌と区別できない可能性があるため、標準的な内視鏡的サーベイランスであることでの腫瘍形成は、多くの場合、8を逃している。良い本は現在避けられないエラーを修復するために消化器を可能にするツールとして、重要な色素増強蛍光イメージングは​​、それによって組織の種類を区別する明確な機能を提供する組織の腺形態を強調しています。?...

最後の40年間で、食道腺癌（EAC）の発生率が大幅に1,2を増加している。まだによる診断の遅れのために、5年生存率が20％未満3。 BE内視鏡サーベイランスの現在の標準は、EACに前駆体は、セグメントのランダムな4-quandrant鉗子」の生検で白色光内視鏡検査である。残念ながら、この技術は、多くの場合、標準白色光画像4に区別するために、平らな微妙で難しいことが異常増殖し、ミス。 生体内で細胞の特徴を強調するために、共焦点レーザー顕微鏡を使用することに成功していたが、病変は依然としてビュー5の減少分野に見逃されることができる。さらに、共焦点microendoscopicイメージングのための領域を強調表示することができます「ブリッジ」の技術を持つことは著しく価値がある。 そのためタージェする能力を改善、強化された赤フラグイメージングモダリティBE中のT生検早期異常増殖が早く、硬化段階でEACの検出に尽力され、そしてより効果的な治療、その後、改善された生存率につながる可能性があります。 VFIは、腺の形態を強調表示し、 インビボ診断6の改善を期待して食道に腫瘍（高度異形成および癌）線引きする、外因性の局所蛍光造影、プロフラビンと高解像度の上皮イメージングを組み合わせた新しい技術である。まもなく適用後の細胞核内に集中するプロフラビンの励起により、蛍光画像は、内視鏡医が腺の形態を視覚化することができ、空間50から100ミクロンの解像度および2.5 CMにまで視野を提供する。結果として、このアプローチはobliteraを有する新生物、BEとから、連続的な、等間隔腺および全体的に均一な形態を有する古典的バレット化生を区別することを可能に胃腸科腺アーキテクチャのTiONから。ここでは、マルチスペクトル内視鏡と、この新しい技術のプロトコルを記述し、高度異形成および癌、良性上皮化生の形態変換を描いたこのデバイスの有用性を実証するための代表的な結果を提供する。

<table border="0" cellpadding="0" cellspacing="0" width="650">
	<tbody>
		<tr>
			<td>Filter*</td>
			<td>Schott North America, Inc., Duryea, Pennsylvania</td>
			<td>Not Applicable</td>
			<td>495-nm long-pass filter</td>
		</tr>
		<tr>
			<td>Halo Cap &#8211; Medium*</td>
			<td>Barrx Medical</td>
			<td>CP-002A</td>
			<td></td>
		</tr>
		<tr>
			<td>Processor*</td>
			<td>Pentax</td>
			<td>EPK-i</td>
			<td></td>
		</tr>
		<tr>
			<td>Multispectral Digital Microscope**</td>
			<td>Not Applicable</td>
			<td>Not Applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark</td>
			<td>KimTech Science</td>
			<td>S-8115</td>
		</tr>
		<tr>
			<td>Cidex</td>
			<td>Advanced Sterilization Products&#160;</td>
			<td>CIDEX OPA Solution</td>
			<td></td>
		</tr>
		<tr>
			<td>Proflavine hemisulfate (0.01% w/v)</td>
			<td></td>
			<td></td>
			<td>FDA (IND 102,217)</td>
		</tr>
		<tr>
			<td>Laser Diode*</td>
			<td>Nichia Corporation</td>
			<td>Not Applicable</td>
			<td>455-nm</td>
		</tr>
		<tr>
			<td>Image Capture*</td>
			<td>Labview</td>
			<td>Not Applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Spray Catheter</td>
			<td>Olympus</td>
			<td>Not Applicable</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>*Equipment specifics within Reference 6. **Equipment specifics within Reference 7</td>
		</tr>
	</tbody>
</table>

diagnosis of neoplasia in barrett&#8217;s esophagus using vital-dye enhanced fluorescence imaging

両方の組織タイプが平らでのみで白色光イメージングと区別できないことができるように、生体内で異常増殖からバレット食道（BE）に良性の上皮化生を区別する能力は依然として困難である。その結果、腺アーキテクチャに焦点を当てモダリティは、食道下部に良性の上皮からの新生物を区別することは有用であろう。 VFIは良性上皮から高度異形成や癌を描写するために、外因性の局所蛍光造影剤を使用する新しい技術です。具体的には、蛍光画像は、内視鏡医が腺の形態を視覚化することができ、空間50から100ミクロンの解像度および2.5 CMにまで視野を提供する。励起時に、古典的なバレット化生、連続、等間隔腺と全体的に均質な形態として表示されます。対照的に、新生物組織が腺フレームワークの完全閉塞で賑わって表示されます。ここでは、の概要を説明します計測およびこの新技術のプロトコルを列挙。 VFIは、疑わしい組織の腺アーキテクチャと胃腸科を与える一方で、携帯異形成は、この様式で解決することはできません。このように、1は形態学的に、この画像診断法を介して低グレード異形成とあるからバレット化生を区別することはできません。視野のより大きな視野と分解能の低下をトレードオフすることにより、この撮像システムは、疑わしい病変を標的とすることは非常に生検少なくとも赤色フラグ撮像装置として用いることができる。精度の対策が期待されている場合には、まだ、、VFIは、遠位食道に（高度異形成や癌のいずれかとして定義）腫瘍の診断のための標準的なイメージング技術になることがあります。

図1Bは、正常扁平上皮によって境界に囲まれていない異形成と古典的なバレット食道を示している。末梢に位置し、青い矢印で示されている平らな扁平上皮組織、以降、鈍い蛍光の均質なカバー内には、腺アーキテクチャに存在している。緑の矢印は、扁平上皮組織を取り巻く円形の緑の線を示している。このアウトラインは、内視鏡のキャップに起因するアーティファクトで?...

Watch this Scientific Journal Video about Diagnosis of Neoplasia in BarrettÃ¢â‚¬â„¢s Esophagus using Vital-dye Enhanced Fluorescence Imaging at JoVE.com

Diagnosis of Neoplasia in Barrett&#8217;s Esophagus using Vital-dye Enhanced Fluorescence Imaging

バイタル色素増強蛍光イメージング（VFI）は腺の形態を強調表示し、食道に腫瘍（高度異形成および癌）線引きする外因性の局所蛍光造影で高解像度の上皮イメージングを組み合わせたin vivoでの技術では新規である。

バイタル色素増強蛍光イメージングを使用して、バレット食道腫瘍の診断

The ability to differentiate benign metaplasia in Barrett&#8217;s Esophagus (BE) from neoplasia in vivo remains difficult as both tissue types can be flat ...

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Unit 1

Disorders of the Esophagus

SCIENTISTS IN ACTION

11.9K ビュー.  Icahn School of Medicine at Mount Sinai. バイタル色素増強蛍光イメージング（VFI）は腺の形態を強調表示し、食道に腫瘍（高度異形成および癌）線引きする外因性の局所蛍光造影で高解像度の上皮イメージングを組み合わせたin vivoでの技術では新規である。 

ビデオ: Diagnosis of Neoplasia in Barrett’s Esophagus using Vital-dye Enhanced Fluorescence Imaging

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三次元光学分解能光音響顕微鏡

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

生物工学

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

生細胞における分子ローターの蛍光寿命イメージング

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

RootChipを使用して、ルート環境の急激な操作によるシロイヌナズナの根の成長のタイムラプス蛍光イメージング

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

単一分子蛍光顕微鏡による受容体ダイナミクスの高分解能時空間解析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

細胞内小胞の追跡への応用：変形二光子顕微鏡での3d軌道の追跡

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

商業顕微鏡でのライブ細胞膜上の分子拡散法への高速蛍光イメージングから

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

1 蛍光顕微鏡を用いた複数の画像モードを実施

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

回転ディスク全内部反射蛍光顕微鏡による細胞の接着の形成の可視化詳細

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Using Nanoplasmon-Enhanced Scattering and Low-Magnification Microscope Imaging to Quantify Tumor-Derived Exosomes

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes have the potential to serve as biomarkers for disease diagnosis and to monitor disease progression and treatment response. However, most exosome analysis procedures require exosome isolation and purification steps, which are usually time-consuming and labor-intensive, and thus of limited utility in clinical settings. This report describes a rapid procedure to analyze specific biomarkers on the outer membrane of exosomes without requiring separate isolation and purification steps. In this method, exosomes are captured on the surface of a slide by exosome-specific antibodies and then hybridized with nanoparticle-conjugated antibody probes specific to a disease. After hybridization, the abundance of the target exosome population is determined by analyzing low-magnification dark-field microscope (LMDFM) images of the bound nanoparticles. This approach can be easily adopted for research and clinical use to analyze membrane-associated exosome biomarkers linked to disease.

Clinical translation of exosome-derived biomarkers for diseased and malignant cells is hindered by the lack of rapid and accurate quantification methods. This report describes the use of low-magnification dark-field microscope images to quantify specific exosome subtypes in small volume serum or plasma samples.

Nanoplasmon の散乱と低倍率の顕微鏡イメージングによる腫瘍由来エキソソームの定量化

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes ...

Contrast-Enhanced Subharmonic Aided Pressure Estimation (SHAPE) Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound imaging are gas-filled, encapsulated microbubbles (diameter &#60; 10 &#956;m) that traverse the entire vasculature and enhance signals by up to 30 dB. These microbubbles also produce nonlinear oscillations at frequencies ranging from the subharmonic (half of the transmit frequency) to higher harmonics. The subharmonic amplitude has an inverse linear relationship with the ambient hydrostatic pressure. Here an ultrasound system capable of performing real-time, subharmonic aided pressure estimation (SHAPE) is presented. During ultrasound contrast agent infusion, an algorithm for optimizing acoustic outputs is activated. Following this calibration, subharmonic microbubble signals (i.e., SHAPE) have the highest sensitivity to pressure changes and can be used to noninvasively quantify pressure. The utility of the SHAPE procedure for identifying portal hypertension in the liver is the emphasis here, but the technique has applicability across many clinical scenarios.

Contrast-Enhanced Subharmonic Aided Pressure Estimation SHAPE Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

A protocol for noninvasively estimating ambient pressures utilizing subharmonic ultrasound imaging of infused contrast microbubbles (following appropriate calibration) is described with examples from human patients with chronic liver disease.

門脈圧亢進症の同定に着目した超音波画像を用いた造影補助圧力推定(SHAPE)

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound ...

Methods Article

バイタル色素増強蛍光イメージングを使用して、バレット食道腫瘍の診断