我々は、C57BL / 6、B1-8 GFPトランスジェニックマウスを提供するためのストライプ化照明手法、K. RajewskyとA.ハーバーマンの開発中に実りある議論をR. Heintzmannに感謝します。私たちは、経済的支援のために（RNに）助成金Rahel-ハーシュフェローシップの下シャリテ、（AEH）はHA5354/4-1とSFB633、プロジェクトA15、（RN）の助成金NI1167/3-1の下でドイツ学術振興を認める。私たちは、特に有益な議論やインフラをサポートするためにネットワークJIMIを認める

<ol>
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フォルカー·アンドレセンはLaVisionバイオテック社、ビーレフェルト、ドイツの株主である。他の著者は、利益相反を持っていない。

生体内光学的イメージングの目的は、動的におよび機能的に健康と病気5における組織および器官の機能を理解するために、細胞運動性および相互作用を可視化することである。その空間分解能、コントラスト並びにその時間分解能を制限し、これを達成するための最も強力な技術、多光子レーザ走査顕微鏡法、依然として前方歪み波に関連する制限を克服しなければならず、散乱、?...

二光子レーザー走査顕微鏡（TPLSM）、赤外線、超短パルス励起1に関連の深い組織のイメージングのための利点で、様々の運動性との相互作用のパターンを明らかにすることによって、単一細胞レベルでの生命過程に関する我々の見解に革命をもたらしました動物の2-5を生きた細胞サブセット。しかし、現在の技術は、それが健康と病気における組織内の細胞応答の分子基盤に関する唯一のまばらな情報をレンダリングするので、新たな治療戦略を開発するための前提条件として臓器機能と機能障害のメカニズムを解明するために、まだ不十分である。現在の技術は、散乱による数百ミクロンの唯一の空間的な窓を可能にし、空間分解能と成体動物の非常にコンパクトな組織で特に明白である信号対雑音比6、フロント歪みの影響を振る。これらの散乱や波面歪みの影響は非常に異質ANによるものである3D空間分解能の深さに依存する劣化への第一歩でも有数の組織における屈折率のD異方性分布し、最終的に弾道励起光子、信号対雑音比、すなわち喪失に起因する信号の全損失に。生命科学や生物医学深部組織用途の観点では、これは現在の技術は、信号対雑音比の低下は、システムがいくつかを見落とす原因となるのに対して乏しい分解能は偽陽性の相互作用をもたらすので一概にセルラー通信を明らかにすることができないことを意味する薄暗い構造間の相互作用。 明白に動的な方法で細胞間相互作用を検出するために、非常に改善された空間分解能が深部組織内で必要とされる。第一励起状態の枯渇で現在導入された強力な特殊な数値計算アルゴリズムに基づいてナノスコピー技術、 例えば構造照明のアプローチ、 例えば 、 </em&gt; STED、RESOLFT、または分子の局在について、 例えば dSTORM、Palmは、固定された細胞にだけでなく、生きた細胞培養7で多くのアプリケーションを発見した。しかし、組織切片、生体組織や生物にこれらのアプリケーションを拡張するために、我々はまだ深刻な技術的な問題を克服する必要があります。二光子励起は、異なる波長で、並びに励起用の単一波長（sw2PE-STED）でSTEDおよび発光が同じで、それぞれ埋め込 ​​まれた細胞9を用いて脳切片8または人工マトリックス中に横方向の解像度を向上させるために適用された刺激標準TPLSMとして距離分解能。 1光子STEDを使用して、樹状突起棘のダイナミクスは、67〜10nm程度の分解能で生きTHY1 EGFPマウスの脳皮質（最大10から15ミクロンの深さ）の表面に画像化することができた。発生生物学のための多目的なツールは2倍に改善された2Dを提供し、多焦点構造化照明顕微鏡によって提供されている解像度。しかしながら、この技術は、例えば、ゼブラフィッシュ胚11と光散乱の低い傾向を有する生物において使用することができる。それでも、これらの技術はいずれも、出産後発症を伴う疾患の生物医学および臨床研究のための重要なモデルであり、数百μm、大人の動物の高度に散乱組織に適用することはできません。 点広がり関数（PSF）、すなわち 、回折限界の波面形状を計算するために使用される近似の独立したが、レンズを通して集束した後、光軸（距離分解能）に沿ったPSFの幅よりも少なくとも3倍大きい光軸（横方向の解像度）12に垂直PSF幅。ゼルニケの係数によって定量化異なる次数の前歪みはかなり、特に視神経に沿って、はるかに大きいのPSFにつながる深部組織イメージングに集中電磁波の波面形状を変更し、波13〜15軸A1。したがって、回折法および波面歪み効果の両方が深い組織イメージングの制限要因としての光軸に沿って解像度を指しています。ナノスの技術は、回折限界を打ち消す一方のみに焦点を当て、軸方向分解能と回折波面歪みの影響の両方に対抗してコントラストを向上させる技術は、高解像度の生体内イメージングのために必要とされる。理想的には、この技術は十分な速さで、携帯動態のモニタリングを可能にするためにもである必要があります。 PSF異常とTPLSMにおける補償光学を用いたコントラスト損失のリアルタイム補正が盛んに研究され、過去十13,14,16-18に改善され、したがって、弾道励起のより良い管理につながる現時点で得られる最善の選択であるされています光子は14。まだ、原因ほとんどはフロント補正を振ることに補償光学で使用されるアプローチ反復的であり、彼らは、HA、その小領域ごとに繰り返さなければVEの（数10×10〜2）は、組織における屈折率の高い不均一性のために、取得速度は、細胞運動性および通信を画像化するために必要なよりも著しく低い。また、適応光学における物理的限界はTPLSMはまだ回折によって決定され改善された。 illumination（SPIN）及び検出側（SPADE）上の時間変調の空間変調は、理論的に解像度を向上させるレーザ走査顕微鏡法に適用することが提案されている。生体内イメージングでの実用化はまだ19を実証されていない。 一緒になって、生きている成体動物における深部組織の画像化の解像度を向上させる技術の開発のための高い需要が存在する。本研究では、マルチビームストライプ照明多光子レーザーの走査プロセスを制御することにより、励磁パターンの空間変調を実現缶詰顕微鏡（MB-SI-MPLSM）20。励起ビーム断面が空間的に変調された構造化照明のアプローチに反して、我々は、励起の空間変調を実現するためにのみスキャン処理を用いる。より長い波長に励起を拡張することによって、我々は独立して、光の、高散乱組織（ 例えばリンパ節、自由散乱路47ミクロン6）の深部組織における空間分解能と信号対雑音比の両方を改善することができる我々は検出非線形の信号、 例えば蛍光、第二高調波発生や他の周波数ミキシング現象。 900 nmの最大励起波長でこのアプローチを用いて、我々は、マウスのリンパ節の胚中心における少数の分子の規模で動的に画像細胞タンパク質構造にできる。従って、我々は良好な抗プロービングの過程で濾胞樹状細胞およびB細胞の表面上の抗原搬送ユニット間の相互作用を視覚化することができ二次リンパ器官における免疫応答の間GEN。

<table border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">ATTO590 &#8211; NSH for coupling</td>
			<td style="width:99px;">ATTO Tec, Germany</td>
			<td style="width:119px;">AD-590-35</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">CD21/35 &#8211; Fab fragment &#8211; ATTO590</td>
			<td style="width:99px;">DRFZ, Berlin</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">The coupling reaction was performed in the central lab of our institution.</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">B1-8 Jk-/- EGFP+</td>
			<td style="width:99px;">Prof. Anne Habermann, Yale Univ., CA, US</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">Can be also found at Jackson Laboratories (JAX).</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">EasySep Negative Selection Mouse B Cell Enrichment&#160;</td>
			<td style="width:99px;">StemmCell Technologies, Germany</td>
			<td style="width:119px;">19854</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Polystyren beads (605), 0.2 &#181;m</td>
			<td style="width:99px;">Life Technologies, Germany</td>
			<td style="width:119px;">F8803</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Name of Equipment</td>
			<td style="width:99px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">TriMScope I</td>
			<td style="width:99px;">LaVision Biotec, Bielefeld, Germany</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">OPO (manual version)</td>
			<td style="width:99px;">APE, Berlin, Germany</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Ti:Sa Laser Chameleon Ultra II</td>
			<td style="width:99px;">Coherent, Duisburg, Germany</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">water-immersion objective lens, 20x, NA 0.95, IR, WD 2 mm</td>
			<td style="width:99px;">Olympus Germany, Hamburg, Germany</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
	</tbody>
</table>

highly resolved intravital striped-illumination microscopy of germinal centers

生体深部組織の多光子顕微鏡による細胞の通信を監視することは、健康と病気の太い組織サンプルや臓器内の免疫細胞の運命を理解するための鍵となります。多光子顕微鏡の走査パターンを制御し、適切な数値計算アルゴリズムを適用することによって、我々は、3倍より良い軸方向分解能及び改善された信号対雑音比を達成することを可能にする、ストライプ状照明アプローチを開発し、 すなわちコントラスト、以上で標準の多光子顕微鏡法と比較して、非常にリンパ器官の組織の散乱内100μmの組織の深さ。撮像深度による磁界検出器の使用にわずかに低かったのに対し、取得速度、ならびに光退色および光損傷効果は、標準的な光電子増倍管ベースの手法と同様であった。ストライプ照明アプローチを使用することにより、我々は、二次濾胞樹状細胞上の免疫複合体沈着物の動態を観察することができる̵1;胚中心のいくつかのタンパク質分子のレベルで。

リンパ節における空間分解能効果的な点広がり関数（EPSF）の寸法は、顕微鏡12の空間分解能に対応する。我々は、（CCDカメラを用いて）確立された二光子レーザー走査顕微鏡技術、 すなわち磁界検出TPLSMと比較して我々のMB-SI-TPLSMにより、リンパ節中のコラーゲン繊維の第二高調波発生信号を取得することにより、この3次元関数を測定し、 （光電子増倍管を...

Watch this Scientific Journal Video about Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers at JoVE.com

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

成体マウスのリンパ節における120μmの深さまでの造影による高解像度の生体内イメージングは​​、空間的に多重焦点二光子顕微鏡の励磁パターンを調節することによって達成される。 100μmの深さでは、このように散乱や回折限界を回避し、（横）487 nmおよび551 nmの（軸方向）の解像度を測定した。

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 ...

highly-resolved-intravital-striped-illumination-microscopy-germinal

Research

JoVE Journal

Immunology and Infection

9.9K ビュー.  Leibniz Institute. 成体マウスのリンパ節における120μmの深さまでの造影による高解像度の生体内イメージングは​​、空間的に多重焦点二光子顕微鏡の励磁パターンを調節することによって達成される。 100μmの深さでは、このように散乱や回折限界を回避し、（横）487 nmおよび551 nmの（軸方向）の解像度を測定した。 

ビデオ: Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

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 ...

免疫・感染学

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

生体内ビデオ顕微鏡による組織内好中球腔内クロールを追跡、経内皮移動と走

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

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

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs.
In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection&#160;on hepatic hemodynamics.
This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug&#8217;s effect on inflammation by quantifying its effect on blood flow.

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 of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

マウスの腸間膜静脈内の生体顕微鏡白血球 - 内皮及び血小板 - 白血球の相互作用

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

リアルタイムで麻酔したマウスに生体顕微鏡により腸間膜静脈に好中球および単球のイメージング

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

表面上のヒトノロウイルスの検出のためのスワブサンプリング方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.

We describe a protocol to surgically expose and stabilize the murine submandibular salivary gland for intravital imaging using upright intravital microscopy. This protocol is easily adaptable to other exocrine glands of the head and neck region of mice and other small rodents.

マウス顎下腺唾液腺の直立した生体顕微鏡検査のための準備

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, ...

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. ...