本研究では、INSERM、エクス·マルセイユ大学、CLフォレスによって得られたHFSPOからキャリア開発賞によってサポートされていました。

倫理文：すべての動物実験は、エクス·マルセイユ大学、フランスの制度動物実験委員会によって承認されたガイドラインおよびプロトコルに従って実施しました。 8での女性のC57BL / 6マウス - 古い10週間は、商業的にDécretN°8 87から848、1987年10月19日、パリの規則に従って取得され、処理されていました。 L.を使用して、すべての実験ドノバン LD1S寄生虫は、フランスと欧州連合の法律の生物学的安全性の規制に従って実施しました。 L. 1.マウスの感染ドノバン前鞭毛寄生虫<ol><li> L.のインビトロで維持するために培地を準備しますドノバン前鞭毛の寄生虫、LD1S（MHOM / SD / 62 / 1S-CL2D）。 <ol><li> M199培地を使用してください。 10％ウシ胎児血清（熱不活性化56℃で30分間）とM199培地サプリメント、25mMのHEPES pHが6.9、12 mMの炭酸水素ナトリウム、1mMのGLUtamine、RPMI 16040ビタミンミックス、10 mMの葉酸、100mMのアデノシン、7.6 mMのヘミン、50 U / mlペニシリンおよび50mg / mlストレプトマイシン。 </li></ol></li><li>文化は、25cm 2の換気フラスコ中で26℃で完全M199培地10ml中の寄生虫をLD1S。 </li><li>連続した差動遠心分離工程を使用して、metacyclic形で濃縮される前鞭毛の寄生虫の集団を準備します。 <ol><li>対数中期寄生虫文化を収穫。 hemocytomerを頼りにしています。完全なM199培地10ml中に5×10 5寄生虫/ mlで再懸濁します。彼らは（約5-6日）で後期定常期に達するまで、嫌気的条件で寄生虫の文化を育てます。 </li><li> 、非metacyclic寄生虫をペレット上清を回収し、10分間26℃で2,500×gで再びそれをスピンする26℃で10分間1200×gでLD1S文化をスピン。 注：この最終ペレットは非常metacyclic前鞭毛の寄生虫に富んでいます。 </li><li> 1にペレットを再懸濁PBSの00μlの。 Vと血球計数器を使用して、寄生虫をカウント：25％グルタルアルデヒドで1/100 Vを固定するための小アリコートを削除します。 </li></ol></li><li> PBS中の1×10 100分の7μlに富む-metacyclic寄生虫集団を希釈します。麻酔したマウスの尾静脈への寄生虫懸濁液を注入30 G針を1mlシリンジを使用して（下記参照）。対照動物では、尾静脈に100μlのPBSを注入。 </li></ol> 2.外科的処置<ol><li> 30分間、70％エタノールで殺生消毒スプレーで作業スペースとすべての手術器具を消毒。 </li><li>ケタミン125mgの/ kgであり、PBSで希釈したキシラジンの12.5ミリグラム/ kgのを含む、動物の体重に応じて、麻酔液を調製します。 </li><li>マウスを計量し、腹腔内に30 G針で1mlシリンジを用いて麻酔薬の適切な投与量を注入します。 </li><li>暖かいマウスを保持し、マウスが完全にanestheであることを確認してください先に進む前に、フットパッドをつまんでtized。マウスでの麻酔液の半分量を再注入するごとに60分。 </li><li>マウスの腹部を剃るとVetedineを使用してクリーニングします。腹部の左側の胸郭下の皮膚や筋肉組織の小さな切開を行います。 </li><li>ちょうど開いた領域の下の腹部に湿ったガーゼを置きます。肝臓を露出させ、ガーゼの上に置きます。肝臓上記湿ったガーゼの別の部分を置きます。 </li><li>顕微鏡下で可視化のために肝臓に、シアノアクリレート結合した金属フレームに、150日24時間、週60ミリメートル厚さ2μmのカバーガラスを配置します。 </li><li>濡れたガーゼでマウスの目を保護します。 </li><li>イメージングセッションの後、頚椎脱臼により麻酔した動物を安楽死させます。 </li></ol>肝臓アーキテクチャの3生体顕微鏡イメージング<ol><li>時を備えた倒立型共焦点顕微鏡で生体顕微鏡の実験を行いますhermostatic室、アポクロマート63Xオイルグリセリン浸対物レンズ（NA 1.4）、アルゴン（488 nm）をヘリウムネオン（543ナノメートル、633 nm）のレーザーと青色ダイオード（405 nm）を制御します。 </li><li>目的に肝臓面を下に覆うカバーガラスと顕微鏡のステージ上にマウスを置きます。 29℃にチャンバーの温度を設定します。正弦波の可視化を可能にするために、肝臓の自己蛍光を使用してフォーカスを調整します。 </li><li>血管系を可視化するために、各マウスについて100μlのPBSで希釈した500μgのBSA-Alexaの647の溶液を調製。 30 G針を有する1mLの注射器を使用して麻酔したマウスの尾静脈に静脈内にこの溶液を注入します。 </li><li>肝細胞核を可視化するために、PBS中のヘキスト33342溶液を調製します。腹腔内に30 G針を1mlシリンジを用いて、マウスに8mg / kgでこの溶液を注入します。 </li><li>肝臓の画像に400 Hzで、通常シーケンシャルモード、63X浸対物レンズとスキャナを使用してください細胞核と肝血管系。ヘキスト励起用の405nmの青色ダイオードとBSA-Alexaの647励起用の633 nmのレーザーをオンにします。アルゴン488 nmレーザーをオンにして、肝臓の自己蛍光を可視化するために大規模な買収帯を定義します。 </li></ol>血流速度測定のための肝臓の4生体顕微鏡イメージング<ol><li>顕微鏡（LAS-AFビューアバージョン3.1.0ビルド8587）のLASソフトウェアを開きます。顕微鏡ソフトウェアを開くときに共鳴スキャナモードを選択します。 </li><li>ハードウェアを設定するには、[設定]タブをクリックします。レーザーをクリックして、ヘリウムネオンレーザ633を選択します。設定をクリックして、12ビットの分解能を選択します。 </li><li>取得メニューをクリックします。ビーム経路の設定]ウィンドウで、目的63Xを選択して、100％に633レーザーパワーを設定します。ドロップダウンメニューPM1-3からのAlexa-633を選択することにより、信号利得をアクティブにし、設定したパラメータをオフにします。 </li><li>取得]タブでは、「XYT」買収を選択モード。 1,024×1024でフォーマット幅を設定します。 8,000 Hzで速度を選択します。 3エアリーユニットのピンホールを選択します。双方向モードを選択しないでください3のズーム倍率を選択します。 </li></ol> RBC速度の5定量分析XYT画像を用いた（方法1） <ol><li>ライブモードでの血液の流れを可視化する「ライブ」アイコンをクリックします。関心のある領域を選択し、分析のための特定の血管を選択します。 USBのコントロールパネルの「X」、「Y」とスキャンフィールド回転座標を調整することにより、水平方向の位置に関心のある血管を設定します。 </li><li>興味のある容器がセットされると、「ライブ」モードを停止します。選択し、ウィンドウ、ライン平均= 1、フレーム平均= 1を設定取得モードで1024×256のフォーマット設定の幅を変更するには、画像を取得するために「スタート」の150をクリックし、「スタック」。 </li><li> 「XYT」画像を使用して、RBC速度の定量分析のためのImageJソフトウェアを開きます。 「ファイル」を選択しますImageJの中のタブには、その後、対象のファイルを選択して「開く」をクリックします。 </li><li> ImageJの中の「プラグイン」メニューに移動し、遺伝子座およびバイオフォーマットオプションをインポート]を選択します。このウィンドウで、「hyperstack」などのパラメータを表示するスタックを選択します。 「xyzct &quot;：スタック順序のオプションでは、オプションを選択します。注：これは、企業買収のすべての一連のウィンドウが開きます。 </li><li> ImageJのメニューの「プラグイン」をクリックし、MTrackJオプションを選択します。小さなウィンドウにある[追加]アイコンをクリックし、追跡するために、RBCをクリックしてください。各次のフレームで同じRBCをクリックして、速度を測定する「尺度」アイコンをクリックしてください。 注：生成されたファイルは.exl形式で保存することができます。 </li><li>容器当たり5 RBCの最小値を追跡し、同じ大きさの3つの個々の血管の最小値を解析します。 </li></ol> XTライン画像を用いたRBC速度の6定量分析（方法2） <ol><li>血液Fを可視化する「ライブ」をクリックしますライブモードで低いです。分析のための特定の血管を選択します。水平位置に関​​心のある血管を設定します。興味のある容器がセットされると、「ライブ」モードを停止します。 </li><li> 「XT」スキャンモードを選択し、ラインスキャンに沿った選択された容器の中央管腔を設定します。 1,024×512で設定された書式の幅、「開始」とXTライン画像を取得の速度= 8,000Hz、ライン平均= 32、時間= 512]をクリックします。 </li><li> .lifファイルを開いて、目的のXTラインの画像を開くことによって、RBC速度の定量分析のための縞を生成します。 LAS-AFソフトウェアでは、「実験」を選択.lifを開いて画像を選択します。注：これは、自動的にカイモグラフを開きます。 </li><li>このカイモグラフ上（X軸上に得られたD、）一定の距離を移動する（ダーク反射を示す）粒子ストリークのために必要な（T、Y軸上で得られる）の時間を測定します。ツール「線を引く」とスジに水平線を描画を選択します。距離に注意してください。μmで、画像の下部に結果タブで生成秒単位の時間。 </li><li>カイモグラフから得られた値を使用して、V =距離/時間として速度を計算します。 </li><li> XTの画像と同じ大きさの3つの個々の容器ごとに最低5粒子筋の最小値を定量化します。 </li></ol>

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The authors have nothing to disclose.

マウス肝臓の生体内顕微鏡検査の最近の開発は、in vivoで 、リアルタイム5,9,10における感染に対する生理的応答の調査のための新しい可能性を開きます。臓器血流量は、しばしば多くの疾患において変化している重大な生理学的パラメータです。しかし、生理学的および感染性条件下での肝血行動態の状態が不十分な探求領域のままです。本研究では、以前に脾臓および腫瘍血...

器官特異的血行動態が、任意の哺乳動物の臓器の重要な生理学的特徴です。血流の異常は、炎症の結果および臓器不全1の徴候である可能性があり。したがって、血流組織、構造および機能は、生理学的および病理学的条件下での分析のためのような重要なパラメータを表示されます。一般的に、特定の器官における血流を解析するために使用されている技術は、技術自体の解像限界を含む、いくつかの制限を含む（ 例えば、血流のドップラーイメージング）当たりの血液のみ（容積絶対血流を測定するための能力臓器を提供するユニット）（ 例えば、光コヒーレンストモグラフィー）および血管2,3の大規模かつ異種集団における速度の平均変化を測定します。肝臓の循環系は、それらのサイズ、構造と機能に不均質である別の容器サブタイプをリンクします。でこの研究は、生体顕微鏡（IVM）イメージング技術は、肝臓器官を構成する個々の血管の特性を明らかにするために、リアルタイムで、高解像度でかつ並列に、インビボでの肝血行動態を評価するために適用されます。この強力な光イメージング技術の最近の開発は、研究者は、高い空間分解能と時間分解能で生きている動物への動的なデータを収集することができます。直接可視化およびインビボでの特異的かつ急速な生物学的過程のリアルタイムの監視を可能にすることにより、IVMは、画像の個々の血管を研究するユニークな機会を提供し、具体的に選択された内の単一の赤血球（RBC）の速度を測定し、定量化します肝臓の血管。 本研究では、肝臓の血行動態上の肝親和性リーシュマニア寄生虫によるマウスの感染の影響を調査するために、マウスの肝臓でのIVMの技術を実装している。L.ドノバンエージェントは、内臓リーシュマニア症、急性·オン·慢性炎症反応と脾臓および肝臓を含む複数の器官で存在する病理学によって特徴付けられる重篤な疾患の原因です。内臓リーシュマニア症の実験マウスモデルでは、脾臓の感染は4プログレッシブであるのに対し、肝臓感染症は、自己解決である。個々の器官に対してリーシュマニア感染のこれらの成果は、まだ完全には理解されていません。病理学的条件の下で、肝臓と脾臓血行動態の検討は、ホスト寄生虫相互作用および疾患の病因に新たな光を当てるします。 我々の実験モデル系は、肝intravasculatureの標識に特異的な蛍光色素の静脈内注射を受け、麻酔したマウスの肝臓を露出し画像化に基づいています。肝臓は、イントラ不可欠な顕微鏡検査のための有利な器官です。小さなincisioを行った後nは腹部に、肝臓を穏やかに外部化され、その後、心拍や呼吸による任意のモーションアーチファクトを低減することを目的としたカバースリップ上に、濡れたガーゼの上に置きました。肝臓は、その後、顕微鏡レンズの視野内に配置されます。 IVMの研究のための2光子顕微鏡を使用する必要が脾臓およびリンパ節と比較して、肝臓の利点は、最大侵入深さで、従来の共焦点顕微鏡の使用を可能にする、その均質な3Dアーキテクチャ/解剖学であります約50μm、生体顕微鏡イメージング5-8。 この研究は、個々の血管におけるRBC速度及び血流速度の定量的測定のための2つの独立した画像化方法を記載しています。第1の方法では、肝血流量は、時間の経過とともに、XY二次元モードを使用して取得されます。得られたXYTデータは個々をの追跡を可能にする無料のImageJソフトウェアでMtrackJプラグインを用いて分析されていますUAL赤血球経時。第2の方法では、単一の血管を選択し、対応する血液の流れは、共焦点レーザー走査顕微鏡のライン走査高速取得モードを使用して分析されます。興味のある容器は、軸線を通って、その中心軸に沿って高頻度で走査されます。血流速度は、次いで、標識されていない暗い赤血球および蛍光標識されたプラズマとの間のコントラストの差に基づいて定量化されます。ラインスキャンに沿って取得された赤血球と血漿の蛍光強度は、角度は、個々のRBCの速度に比例し、筋を得るために時間に対してプロットされています。 この記事の目的は、イメージングのための簡単​​かつ再現性のある方法を提供し、肝臓の個々の血管内の血流速度を測定し、マウスの手術、IVMとの速度の定量分析の成功のパフォーマンスのための基本的なツールを利用できるようにすることです個々の赤血球。 T彼のアプローチは、研究者は、病理学的条件下での血流速度に新たな洞察を得ることができます。

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intravital microscopy imaging of the liver following leishmania infection: an assessment of hepatic hemodynamics

生体顕微鏡（IVM）は、リアルタイムで、ライブ動物における様々な生物学的事象の可視化、監視および定量化を可能にした強力な光イメージング技術です。この技術は、非常に生理的プロセスおよび特定の器官中の病原体媒介性の現象の理解を進めています。 本研究では、IVMはマウス肝臓に適用され、プロトコルが設計されている画像に、インビボで肝臓の循環系および個々の肝臓の血管内の赤血球（RBC）の速度を測定します。肝臓器官を特徴付ける別の容器サブタイプを視覚化し、血流速度測定を実行するために、C57BL / 6マウスに、静脈内に、肝臓に関連する血管系を標識する蛍光プラズマ試薬を注射します。 IVMは、in vivo、リアルタイム、関心のある特定の容器内のRBC速度の測定に有効になります。この方法論を確立することが可能になります生理学的および病理学的条件下での肝血行動態を調査します。最終的には、このイメージングベースの方法論は、Lの影響を研究するために重要になります肝血行動態上のドノバン感染。 この方法は、他の感染モデルとマウスの臓器に適用することができ、さらに血流に及ぼす影響を定量化することによって、炎症に対する薬物の効果を試験する臨床を事前に拡張することがあります。

肝臓での正弦波の特定の建築組織が ​​可視化され、この器官（ 図1、パネルBおよびC、緑）、肝細胞核の標識のためのヘキストの腹腔内注射の自己蛍光性に基づくことができる（ 図1B、青）および肝循環系（ 図1C、赤）の染色用蛍光BSAの静脈内注射。肝臓は、直径（D）小血管の（Dで40-700μmの2の範囲の異なる機能や構造や大きさで、いくつかの...

Watch this Scientific Journal Video about Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics at JoVE.com

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

以下の肝臓の生体顕微鏡イメージング<em&gt;リーシュマニア</em&gt;感染症：肝血行動態の評価

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

intravital-microscopy-imaging-liver-following-leishmania-infection-an

Research

JoVE Journal

Immunology and Infection

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

11.4K ビュー.  University of Aix-Marseille. This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

ビデオ: Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

ライブホストのトランスジェニックリーシュマニア原虫のin vivoイメージング

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

免疫・感染学

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

鼠径リンパ節の生体顕微鏡

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

2光子顕微鏡を用いてマウスの胸腺の生体内イメージング

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

脾臓の生体顕微鏡検査：寄生虫モビリティと血流​​の定量的解析

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

マウス膝窩リンパ節の生体内イメージング

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

胚中心の高解像度生体内ストライプ照明顕微鏡

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

せん断応力のヒト肝類洞内皮条件のことを白血球の募集を研究するフロー接着アッセイ

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

CXCR6.Gfpレポーターマウスを使用して、健康で罹患肝臓における免疫細胞の長期生体内多光子顕微鏡イメージング

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

リポ多糖投与マウス肝臓における免疫応答時に白血球の二光子励起生体イメージング

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

イメージング 2 光子生体顕微鏡を用いたインフルエンザ ウイルス感染症の中に気管粘膜の細胞相互作用

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...