This research was supported by NIH-CA100324, Einstein National Cancer Institute's cancer center support grant P30CA013330, R01CA172451 to JWP and the Integrated Imaging Program. This technology was developed in the Gruss-Lipper Biophotonics Center and the Integrated Imaging Program at the Albert Einstein College of Medicine. We acknowledge the support of these Centers in this work. The authors thank Mike Rottenkolber, Ricardo Ibagon and Anthony Leggiadro of the Einstein machine shop for their skilled and timely craftsmanship, the laboratory of Matthew Krummel for generously sharing their window design drawings, Kevin Elicieri and Jeremy Bredfeldt for their expertise in microscopy and their amplifier recommendations and Allison Harney and Bojana Gligorijevic for informative discussions.

The authors have nothing to disclose.

例えば、タンパク質や抗体などの蛍光標識された機能タグと組み合わせて、インビボ光学イメージングにおける高分解能が劇的に転移カスケードの我々の理解を増加しています。これは、直接可視化し、単一細胞と腫瘍細胞における小細胞パラメーター、宿主細胞およびそれらの微小環境の定量化を可能にしました。原発腫瘍内のこのイメージングは、例えば、成長の浸潤や播?...

高分解能生体内光学イメージングは​​、単一細胞および細胞下のパラメータを測定し、定量化することを可能にする、多くの生物学的プロセスを理解するために重要であることが証明されています。癌研究では、腫瘍細胞および間質細胞の生体内イメージングは、無傷の動物にのみ存在している多くの微小環境の相互作用6-11の発見につながりました。 インビボでの単細胞解像度光学イメージングを用いた乳癌における血管内および腫瘍細胞の播種に関連付けられた微小環境についての発見は、さらに予後および乳癌患者12-16における治療に対する反応のための新規マーカーをもたらしました。無傷の内部の重要な臓器内の深いご覧いただけ最高のイメージング技術は、臓器全体の素晴らしい景色を眺めることができますし、彼らは臨床症状を引き起こす前であっても病理を明らかにすることができ、臨床的モダリティ（MRI、PET、CT）です。彼らは、Hができませんowever、転移の初期段階起因単細胞解像度の欠如に腫瘍の進行を駆動する細胞メカニズムを明らかにします。時間によって肺転移は、これらのモダリティに表示され、それらは十分に確立し、増殖しています。肺に到着播種性腫瘍細胞の90％が17を生き残るか、最初は18休止状態のまま、彼らははるかに早く、以前に19を予想より到着観察は、到着と生存の最も初期の段階を画像化するために重要となりませんいずれかのことを推定を考えます離れた部位に転移性播種し、腫瘍増殖の再発のプロセスを理解します。 肺にこれらの観測を実行することが極めて困難であることが判明しました。イメージング研究の大半は、単一の時点で肺にビューを提供エクスビボまたは外植調剤20-23を 、利用してきました。これらの製剤は、有用な情報を提供されますがrmation、彼らは微小環境のさまざまなコンポーネント間で発生する相互作用、因果関係、およびダイナミクスの完全な理解を与えることはありません。適切な循環系（およびホメオスタシスの同時アンバランス）と身体の免疫系の残りの部分から切断の欠如は、それが望ましい、これらの製剤は、 生体内での無傷組織に発生結論を検証することができます。 多くのグループがWearnとドイツが胸膜層24を露出させる外科的に最初とテリー移植可能な観察窓25を利用することが第一であると無傷の肺2,4,5,24-33の生体内イメージングを行いました。 肺の高分解能イメージングが大幅に肺の一定の動きにより阻害され、いくつかの技術が、この制限を克服するために開発されてきました。ワーグナーやフィレー27は、イヌの肺の自然な動きを研究しましたそして、ワーグナーが組織28を固定するために彼のウィンドウ外科手術の準備に真空を利用しながら、相対的に静止領域の上に彼らの移植ウィンドウを見つけるために彼らの外科的プロトコルを設計しました。気管支クランプ、シーケンシャル無呼吸と同期撮影、オーバーサンプリング取得、肺葉と真空34の接着：その時以来、種々の技術は、画像に含めて肺を利用されてきました。これらの各々は、長所と短所があり、誰の技術は、他の34にとして優れ出現していません。例えば、気管支クランプとシーケンシャル無呼吸は、肺内のガスの正常な交換を変更し、無気肺を引き起こす可能性があります。同期撮影とオーバーサンプリングされた買収は、これらの欠点を有するが、高速や特殊な画像機器に広くアクセスすることはできません必要はありません。最後に、肺の両方の接着と真空技術は、上述した欠点の両方を回避するが、注意が徳でない場合は、せん断力誘発される損傷を示すことができますアン。近年では、真空窓が小型化されたマウスでの使用に適合共焦点及び多光子顕微鏡4,5,33を使用して、優れた高解像度イメージングは2達成された。 表1は、この歴史を要約し、小説を記述するそれら書類を強調生体肺の撮像ウインドウの使用の進歩。 このプロトコルは、可能な限り最高の細胞下解像度でのライブ、無傷の肺の画像の転移に長時間タイムラプス多光子生体顕微鏡の使用を記載しています。画像は、高開口数の対物レンズと、複数の光電子増倍管（PMT）検出器を装備した多光子顕微鏡を用いて、最大12時間に取得されています。トランスジェニックマウスモデルは、血管系および腫瘍細胞を標識する蛍光高分子量デキストラン及び蛍光タンパク質トランスフェクトされた腫瘍細胞（と共に蛍光標識ネイティブマクロファージに利用さrespectivelY）。蛍光標識された細胞のこの選択は、腫瘍細胞 - 内皮細胞 - マクロファージの相互作用とダイナミクスの可視化を可能にしているが、このプロトコルは、蛍光または非蛍光マウスの任意の株のために動作します。買収後、残留ドリフト運動（もしあれば）フィジープラグイン35,36およびカスタムマクロの時間平均に標識されていない循環血液細胞によって引き起こさ点滅排除するための脈管を用いて除去されます。 このプロトコルは、撮像転移に焦点を当てているが、技術は、肺における高解像度の単一細胞イメージングで観察多くの他の生物学的プロセスにも適用可能です。

<table><tbody><tr><td>Nickel-Plated Brass Vacuum Regulator 1/8 NPT Female, w/ Gauge, 0 - 20&quot; Hg Vacuum</td><td>McMaster Carr</td><td>4172K12&amp;nbsp;</td><td>Vacuum Regulator</td></tr><tr><td>Brass Barbed Hose Fitting Adapter for 1/4&quot; Hose ID X 1/8&quot; NPTF Male Pipe</td><td>McMaster Carr</td><td>5346K13</td><td>Vacuum Regulator Hose Adapter</td></tr><tr><td>Pyrex Brand Filtering Flasks with Tubulation; Neck tooled for rubber stopper No. 4; Capacity: 50 ml</td><td>Corning Life Sciences Glass</td><td>5360-50</td><td>Vacuum Flask</td></tr><tr><td>Round Glass Coverslips Thickness #1.5, 0.16 - 0.19 mm 10 mm dia.&amp;nbsp;</td><td>Ted Pella, Inc.</td><td>260368</td><td>Cover slips</td></tr><tr><td>Exel International Disposable Safelet I.V. Catheters; 22 g x 1 in.&amp;nbsp;</td><td>Exel International</td><td>26746</td><td>Tracheal Catheter</td></tr><tr><td>PERMA-HAND Black Braided Silk Sutures, ETHICON LIGAPAK Dispensing Reel Size 2-0</td><td>VWR</td><td>95056-992</td><td>String</td></tr><tr><td>Liquid Super Glue, Clear, 0.14 oz</td><td>Hendel Corp.</td><td>LOC1647358</td><td>Cyano-acrylate Glue</td></tr><tr><td>Tetramethylrhodamine isothiocyanate&amp;ndash;Dextran</td><td>Sigma-Aldrich</td><td>T1287-500MG</td><td>155 kD Dextran</td></tr><tr><td>Laboratory Clear Tygon PVC Tubing, 1/16&quot; ID, 1/8&quot; OD, 1/32&quot; Wall Thickness, 25 ft. Length</td><td>McMaster Carr</td><td>5155T12</td><td>Thin Tubing &amp;amp; Tubing for Luer</td></tr><tr><td>Crack-Resistant Polyethylene Tubing, 1/8&quot; ID, 1/4&quot; OD, 1/16&quot; Wall Thickness, White, 50 ft. Length&amp;nbsp;</td><td>McMaster Carr</td><td>5181K24&amp;nbsp;</td><td>Thick Tubing</td></tr><tr><td>Depillatory Lotion</td><td>Nair</td><td>-</td><td></td></tr><tr><td>Micro Medical Tubing 95 Durometer LDPE</td><td>Scientific Commodities Inc.</td><td>BB31695-PE/1</td><td>Tubing for tail vein catheter</td></tr><tr><td>30 G x 1 in. BD PrecisionGlide Needle</td><td>BD</td><td>305128</td><td>Needles for tail vein catheter</td></tr><tr><td>Puritan Nonsterile Cotton-Tipped Swabs&amp;nbsp;</td><td>Fisher Scientific</td><td>867WCNOGLUE</td><td></td></tr><tr><td>Clear Polycarbonate Barbed Tube Fitting, Reducing Straight for 3/32&quot; x 1/16&quot; Tube ID</td><td>McMaster Carr</td><td>5117k51</td><td>Connectors between tubes</td></tr><tr><td>One-Hole Rubber Stoppers</td><td>Fisher Scientific</td><td>14-135F</td><td>Stopper for Vacuum Flask</td></tr><tr><td>SHARP Precision Barrier Tips, For P-100, 100 &amp;micro;l</td><td>Denville Scientific Inc.</td><td>P1125</td><td>Pipette Tip</td></tr><tr><td>Laboratory tape</td><td>Fisher Scientific</td><td>159015R</td><td></td></tr><tr><td>Puralube</td><td>Henry Schein Animal Health</td><td>008897</td><td>Opthalmic Ointment</td></tr><tr><td>Gemini Cautery Kit</td><td>Harvard Apparatus</td><td>726067</td><td>Cautery Pen</td></tr><tr><td>Graefe Micro Dissecting Forceps; Serrated; Slight Curve; 0.8 mm Tip Width; 4&quot; Length</td><td>Roboz Surgical</td><td>RS-5135&amp;nbsp;</td><td>Forceps</td></tr><tr><td>Extra Fine Micro Dissecting Scissors 4&quot; Straight Sharp/Sharp 24 mm</td><td>Roboz Surgical</td><td>RS-5912</td><td>Sharp Scissors</td></tr><tr><td>Micro Dissecting Scissors 4&quot; Straight Blunt/Blunt</td><td>Roboz Surgical</td><td>RS-5980</td><td>Blunt Scissors</td></tr><tr><td>Wipes</td><td>Fisher Scientific</td><td>06-666-A&amp;nbsp;</td><td>Harness</td></tr><tr><td>PhysioSuite System</td><td>Kent Scientific</td><td>PhysioSuite</td><td>Vitals Monitor</td></tr><tr><td>1 ml Syringe, Tuberculin Slip Tip</td><td>BD</td><td>309659</td><td>Syringe</td></tr><tr><td>Cyano acrylate</td><td>Staples</td><td>LOC1647358</td><td>Cover Slip Adhesive</td></tr><tr><td>Petroleum Jelly</td><td>Fisher Scientific</td><td>19-086291</td><td>Water Barrier</td></tr><tr><td>Adapter Luer Cannulla 1.5 - 2.2 mm</td><td>Harvard Apparatus</td><td>734118</td><td>Catheter Connector</td></tr><tr><td>PhysioSuite MouseSTAT pulse oximeter</td><td>Kent Scientific</td><td></td><td>Pulse Oximeter</td></tr><tr><td>Isoethesia (isoflurane)</td><td>Henry Schein Animal Health</td><td>50033</td><td>250 ml</td></tr><tr><td>Oxygen</td><td>TechAir</td><td>OX TM</td><td></td></tr><tr><td>1x PBS</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>PVC Ball Valve, Push to Connect, 1/4 In</td><td>Grainger</td><td>3CGJ7</td><td>Vacuum Valve</td></tr><tr><td>Small Animal Ventilator</td><td>Harvard Apparatus</td><td>683</td><td>Alternative is available from Kent Scientific: MouseVent</td></tr><tr><td>OptiMEM Reduced Serum Medium</td><td>ThermoFisher Scientific</td><td>31985062&amp;nbsp;</td><td></td></tr><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>ThermoFisher Scientific</td><td>11668019</td><td></td></tr><tr><td>MacBlue Tg(Csf1r*-GAL4/VP16,UAS-ECFP)1Hume/J Mice</td><td>Jackson Laboratory</td><td>026051&amp;nbsp;</td><td></td></tr><tr><td>Multiphoton Microscope</td><td>Olympus</td><td>Fluoview FV1000</td><td>Alternative to custom built scope</td></tr><tr><td>Environmental Enclosure</td><td>Precision Plastics</td><td>Chamber for FV1000</td><td>Alternative to custom built enclosure</td></tr><tr><td>Phosphate Buffered Saline</td><td>ThermoFisher Scientific</td><td>14190136</td><td></td></tr><tr><td>Laser Power Meter</td><td>Coherent</td><td>FieldMaxIITOP</td><td></td></tr><tr><td>Laser Power Meter Head</td><td>Coherent</td><td>PM10</td><td></td></tr><tr><td>pcDNA3-Clover Fluorescent Protein Vector</td><td>Addgene</td><td>40259</td><td></td></tr><tr><td>G418 Sulfate Selective Antibiotic</td><td>ThermoFisher Scientific</td><td>10131027</td><td></td></tr><tr><td>MoFlo Fluorescent-Activate Cell Sorter&amp;nbsp;</td><td>Beckman Coulter</td><td>XDP</td><td></td></tr><tr><td>Trypsin EDTA 1x</td><td>Corning</td><td>25-052-Cl</td><td></td></tr><tr><td>40 &amp;micro;m Mesh</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>96 Well Plate</td><td>Costar</td><td>3599</td><td></td></tr><tr><td>60 mm Culture Dish</td><td>Corning</td><td>430196</td><td></td></tr><tr><td>10 cm Culture Dish</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A4503</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline 1x</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>C57BL/6J Mouse</td><td>Jackson Laboratory</td><td>000664&amp;nbsp;</td><td></td></tr><tr><td>Kim Wipes</td><td>Fisher Scientific</td><td>06-666-A&amp;nbsp;</td><td></td></tr></tbody></table>

long-term high-resolution intravital microscopy in the lung with a vacuum stabilized imaging window

例えば、肺、肝臓や骨などのセカンダリサイトへの転移は約90％1の死亡率との衝撃的な出来事です。これらのサイトのうち、肺が原因ボディ、繊細な自然と適切な生理機能を維持する上で重要な役割内のその囲まれた位置に生体光学イメージングを使用して評価することが最も困難です。臨床モダリティ（陽電子放出断層撮影（PET）、磁気共鳴イメージング（MRI）及びコンピュータ断層撮影（CT））は、この組織の非侵襲的な画像を提供することができるが、それらは、単一のピクセルと、最も早く播種イベントを可視化するために必要な解像度を欠いていますほぼ千の細胞からなります。転移性肺の現行モデルは、単に腫瘍細胞の到着後のイベントは生存とその後の成長のために確定的公準を播種します。これは、単一セルの解像度を2とリアルタイム生体イメージングツールが播種セル画の表現型を定義するために必要とされることを意味しますlsコマンドは、これらのモデルをテストし。肺の高分解能光学的イメージングは、 生体外の様々な製剤を用いて行われてきたが、これらの実験は、典型的には、単一の時間ポイントアッセイであり、アーチファクトと劇的に変化した環境（温度、豊富、サイトカインなどによる誤結論に影響されやすいです）胸腔および循環系3からの除去に起因します。最近の研究は、無傷の肺のタイムラプス生体光イメージングを示した真空を使用して可能であるが、撮像窓2,4,5を安定化し、典型的なイメージング時間は約6時間に限られていました。ここでは、12時間の期間にわたって、このようなウィンドウを利用し、肺の長期生体内タイムラプスイメージングを行うためのプロトコルを記述します。タイムラプス画像シーケンスは、この方法を用いて得られた細胞間相互作用、膜動態および肺における血管灌流の可視化および定量化を可能にします。我々はさらにD肺微小血管系の従来にないクリアな視界を提供する画像処理技術を傍接させます。

この方法を用いて達成することができる結果の種類を示すために、我々は、手術前の時点を変えることでMacBlueマウス44の尾静脈に蛍光タンパク質で標識されたクローバーE0771-LG腫瘍細胞を注射しました。手術後、155 kDのローダミン標識デキストランは、脈管構造とタイムラプス撮影を行ったマークするために静脈内注射しました。 <p class="jove_content" fo:keep-to...

Watch this Scientific Journal Video about Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window at JoVE.com

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

真空安定化撮像窓と肺における長期高分解能生体顕微鏡

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

long-term-high-resolution-intravital-microscopy-lung-with-vacuum

Research

JoVE Journal

Cancer Research

13.1K ビュー.  Albert Einstein College of Medicine. This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window. 

ビデオ: Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

Ex Vivoひと肺腫瘍内常駐 cd8 陽性 T リンパ球の画像スライス共焦点顕微鏡を用いた

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

がん研究

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

2光子顕微鏡を用いて感染肺における好中球による貪食とNET -形成の直接観察

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

免疫・感染学

Longitudinal Intravital Imaging Through Clear Silicone Windows

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging windows is particularly powerful because it allows longitudinal observation of the same tissue over days to weeks. Typical imaging windows comprise a glass coverslip in a biocompatible metal frame sutured to the mouse&#8217;s skin. These windows can interfere with the free movement of the mice, elicit a strong inflammatory response, and fail due to broken glass or torn sutures, any of which may necessitate euthanasia. To address these issues, windows for long-term abdominal organ and mammary gland imaging&#160;were developed from a thin film of polydimethylsiloxane (PDMS), an optically clear silicone polymer previously used for cranial imaging windows. These windows can be glued directly to the tissues, reducing the time needed for insertion. PDMS is flexible, contributing to its durability in mice over time&#8212;up to 35 days have been tested. Longitudinal imaging is imaging of the same tissue region during separate sessions. A stainless-steel grid was embedded within the windows to localize the same region, allowing the visualization of dynamic processes (like mammary gland involution) at the same locations, days apart. This silicone window also allowed monitoring of single disseminated cancer cells developing into micro-metastases over time. The silicone windows used in this study are simpler to insert than metal-framed glass windows and cause limited inflammation of the imaged tissues. Moreover, embedded grids allow for straightforward tracking of the same tissue region in repeated imaging sessions.

An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin.&#160;These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals.

透明なシリコーン窓による縦方向の生体内イメージング

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging ...

A Permanent Window for Investigating Cancer Metastasis to the Lung

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as the bone, brain, and lung. Although extensively studied, the mechanistic details of this process remain poorly understood. While common imaging modalities, including computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI), offer varying degrees of gross visualization, each lacks the temporal and spatial resolution necessary to detect the dynamics of individual tumor cells. To address this, numerous techniques have been described for intravital imaging of common metastatic sites. Of these sites, the lung has proven especially challenging to access for intravital imaging owing to its delicacy and critical role in sustaining life. Although several approaches have previously been described for single-cell intravital imaging of the intact lung, all involve highly invasive and terminal procedures, limiting the maximum possible imaging duration to 6-12 h. Described here is an improved technique for the permanent implantation of a minimally invasive thoracic optical Window for High-Resolution Imaging of the Lung (WHRIL). Combined with an adapted approach to microcartography, the innovative optical window facilitates serial intravital imaging of the intact lung at single-cell resolution across multiple imaging sessions and spanning multiple weeks. Given the unprecedented duration of time over which imaging data can be gathered, the WHRIL can facilitate the accelerated discovery of the dynamic mechanisms underlying metastatic progression and numerous additional biologic processes within the lung.

Here, we present a protocol for the surgical implantation of a permanently indwelling optical window for the murine thorax, which enables high-resolution, intravital imaging of the lung. The permanence of the window makes it well-suited to the study of dynamic cellular processes in the lung, especially those that are slowly evolving, such as metastatic progression of disseminated tumor cells.

肺への癌転移を調査するための永久的な窓

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

腫瘍関連血管系を使用しての生体顕微鏡が背高度な蛍光トランスジェニック マウスの皮下脂肪のウィンドウの部屋

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

医学

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

In Vivo 光シート顕微鏡観察に基づくアレルギー性炎症の実験的肺ホーミング ヒトリンパ球のイメージング高度な

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an ...

Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and other respiratory pathologies in vivo is difficult due to the limited accessibility and inherent motion artifacts of the lungs. Nonetheless, various approaches have been developed to overcome these challenges. This protocol describes a method for intravital fluorescence microscopy to study real-time leukocyte-endothelial interactions in the pulmonary microcirculation in an experimental model of ALI. An in vivo lung imaging system and 3-D printed intravital microscopy platform are used to secure the anesthetized mouse and stabilize the lung while minimizing confounding lung injury. Following preparation, widefield fluorescence microscopy is used to study leukocyte adhesion, leukocyte rolling, and capillary function. While the protocol presented here focuses on imaging in an acute model of inflammatory lung disease, it may also be adapted to study other pathological and physiological processes in the lung.

Intravital fluorescence microscopy can be utilized to study leukocyte-endothelial interactions and capillary perfusion in real-time. This protocol describes methods to image and quantify these parameters in the pulmonary microcirculation using a vacuum-stabilized lung imaging system.

真空安定化イメージングシステムを用いた実験的急性肺損傷における肺微小循環の生体内広視野蛍光顕微鏡

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung ...

<meta charset="utf8"></head><body>マウス肺移植における細胞動態のリアルタイムモニタリング

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in na&#239;ve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

<meta charset="utf8"></head><body>著者スポットライト:Intravital Imagingによる肺移植免疫学の進歩

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

移植されたマウス肺の生体内2光子顕微鏡検査

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by ...

真空安定化撮像窓と肺における長期高分解能生体顕微鏡

要約

さらに動画を探す

この動画の章

Ex Vivoひと肺腫瘍内常駐 cd8 陽性 T リンパ球の画像スライス共焦点顕微鏡を用いた

2光子顕微鏡を用いて感染肺における好中球による貪食とNET -形成の直接観察

透明なシリコーン窓による縦方向の生体内イメージング

肺への癌転移を調査するための永久的な窓

腫瘍関連血管系を使用しての生体顕微鏡が背高度な蛍光トランスジェニックマウスの皮下脂肪のウィンドウの部屋

In Vivo 光シート顕微鏡観察に基づくアレルギー性炎症の実験的肺ホーミングヒトリンパ球のイメージング高度な

真空安定化イメージングシステムを用いた実験的急性肺損傷における肺微小循環の生体内広視野蛍光顕微鏡

移植されたマウス肺の生体内2光子顕微鏡検査

移植されたマウス肺の生体内2光子顕微鏡検査

移植されたマウス肺の生体内2光子顕微鏡検査

真空安定化撮像窓と肺における長期高分解能生体顕微鏡

要約

さらに動画を探す

この動画の章

Ex Vivoひと肺腫瘍内常駐 cd8 陽性 T リンパ球の画像スライス共焦点顕微鏡を用いた

2光子顕微鏡を用いて感染肺における好中球による貪食とNET -形成の直接観察

透明なシリコーン窓による縦方向の生体内イメージング

肺への癌転移を調査するための永久的な窓

腫瘍関連血管系を使用しての生体顕微鏡が背高度な蛍光トランスジェニック マウスの皮下脂肪のウィンドウの部屋

In Vivo 光シート顕微鏡観察に基づくアレルギー性炎症の実験的肺ホーミング ヒトリンパ球のイメージング高度な

真空安定化イメージングシステムを用いた実験的急性肺損傷における肺微小循環の生体内広視野蛍光顕微鏡

移植されたマウス肺の生体内2光子顕微鏡検査

移植されたマウス肺の生体内2光子顕微鏡検査

移植されたマウス肺の生体内2光子顕微鏡検査

腫瘍関連血管系を使用しての生体顕微鏡が背高度な蛍光トランスジェニックマウスの皮下脂肪のウィンドウの部屋

In Vivo 光シート顕微鏡観察に基づくアレルギー性炎症の実験的肺ホーミングヒトリンパ球のイメージング高度な