This work was supported by the Deutsche Forschungsgemeinschaft (DU 1190/3-1).

Alle Tierversuche wurden in Übereinstimmung mit dem Deutschen Tierschutzgesetz (TierSchG) durchgeführt. Die Mäuse wurden untergebracht ist und in Übereinstimmung mit der guten Tierpraxis gehandhabt wie FELASA (www.felasa.eu/guidelines.php) und der nationalen Tierschutzkörper GV-SOLAS (www.gv-solas.de/index.html) definiert. Die Tierschutzkomitee der Universität Freiburg sowie die lokalen Behörden (Regierungspräsidium Freiburg) stimmten allen Tierversuchen. 1. Tier Handhabung, Vorbereitung und Induktion der Entzündung <ol><li> Verwenden Sie 4 - 6 Wochen alte männliche Mäuse mit einem Gewichtsbereich von 16 bis 20 g. Hinweis: Wenn die Mäuse älter sind oder schwerer präsentieren sie überschüssiges Fett rund um das Schiff, die Begrenzung der mikroskopischen Beobachtung. </li><li> Sterilisieren Sie alle Werkzeuge und Mikroskoptisch vor der Operation. </li><li> Injizieren Sie 20 mg / kg LPS (Lipopolysaccharid) intraperitoneal 4 Stunden vor der in den Wohn Maus Mikroskopie, eine bakterielle Inflamm induzierenation. </li><li> Vorwärmen eines 0,9% igen Kochsalzlösung in einem Wasserbad bei 37 ° C, um die Kunststoffkammer und Mesenterium Gewebe zu befeuchten. </li><li> Betäuben die Maus mit einer intraperitonealen Injektion von Ketamin (100 mg / kg) und Xylazin (5 mg / kg) unmittelbar vor der Mikroskopie Verfahren. Alternative Anästhesie verwendet werden kann, beispielsweise 2% Isofluran Einatmen. Überprüfen Sie die ordnungsgemäße Betäubung durch den Verlust der Reaktion auf Reflexstimulation (toe Prise oder Schwanz mit festem Druck). </li><li> Enthaaren den Bauch mit einem Rasierer und entfernen Sie lose Haare mit Gaze mit Ethanol 70% gesättigt. </li><li> Gelten vet Salbe auf die Augen der Maus an Trockenheit zu verhindern, während sie unter Anästhesie. </li></ol> 2. Chirurgie <ol><li> Sterilisieren Sie den Bauch mit 70% Ethanol. Dieses Verfahren ist kein Verfahren und sterile könnte tödliche am Ende des Experiments. </li><li> Führen Sie eine mediane Laparotomie: Öffnen Sie die Bauchhaut mit kleinen, gebogenen Pinzette und eine kleine Schere. Identifizieren Sie die epigastrical Gefäße und öffnen Sie das Peritoneum im Bereich der Linea alba, um die Schiffe zu schützen. </li><li> Geben Sie einige Tropfen von Kochsalzlösung vorgewärmt in die Bauchhöhle, um das Gewebe feucht zu halten. </li><li> Label von Leukozyten und Blutplättchen durch die Injektion fluoreszenz 50 ul Rhodamin 6G (1 mg / ml) retroorbital wie zuvor beschrieben 9,10. </li><li> Exteriorisieren eine Schleife von ileumand Ort in einem Petrischale mit einem Durchmesser von 10 cm und stellen Sie sicher, um das Gewebe feucht durch Anlegen der 37 ° C vorgewärmt halten Kochsalzlösung (0,9%) alle zwei Minuten. </li></ol> 3. Intravitalmikroskopie <ol><li> Platzieren Sie die Maus unter dem Mikroskop und bringen das Gekröse Vene mit einem Durchmesser von 200 bis 300 &amp; mgr; m in der Mitte der Ansicht. Wählen Sie ein Schiff ohne sichtbare Fett Umgebung. </li><li> Berühren Sie nicht die Gekröse Gefäße, wodurch die Stimulation des Endothels zu vermeiden. Behandeln Sie das Ileum Schleife vorsichtig. </li><li> Visualisieren Blutkörperchen-Endothel-Interaktionenmit einem invertierten oder aufrechten Mikroskop und eine Kamera mit einem Mikroskop-Software. Nehmen Blutkörperchen-Endothel-Interaktionen für 1 min in 4 verschiedenen Adern pro Maus. </li><li> Euthanize die Maus durch zervikale Dislokation nach Abschluss der Bildgebung Experimente. </li></ol> 4. Analyse <ol><li> Durchführung der Analyse offline und für alle Parameter geblendet. Durchführung von Analysen unter Verwendung einer geeigneten Software-Programm. </li><li> Bestätigen stabile und inter vergleichbare Blutströmungsverhältnisse in hoher Zeitauflösung cine-Clips (maximale Bildrate) auf intraluminale Blutzellenstrom konzentriert. </li><li> Quantifizierung der Anzahl von Roll Leukozyten. Dazu ziehen Sie eine vertikale Linie durch die Vene und zählen alle Leukozyten Überschreiten dieser Linie in 1 min. </li><li> Bestimmen Sie Walzgeschwindigkeit durch Messen der Zeit eine einzige Leukozyten muss einen Abstand von 50 &amp; mgr; m passieren, während stabil Rollen auf Endothel. Um dies zu tun, ziehen zwei vertikale Linien in einem Abstand von 50 &amp; mgr; turch die Vene. <ol><li> Die Zeit messen, ein Vertreter Leukozyten muss von einer Zeile zur other.Calculate die Geschwindigkeit der Leukozyten durch Teilen 50 um über den Zeitraum (&amp; mgr; m / s) zu erhalten. </li></ol></li><li> Messen Leukozytenadhäsion in einem Bereich von 0,04 mm 2. Dazu zeichnen Sie ein Quadrat mit der Seitenlänge von 200 &amp; mgr; m in die Vene. <ol><li> Zählen Sie die fest haftenden Leukozyten, definiert als ohne sichtbare Bewegung für 30 Sekunden, in diesem Platz. </li></ol></li><li> Zählen Sie die Anzahl von Blutplättchen an einem Leukozyten verpflichtet, Plättchen-Leukozyten-Wechselwirkungen zu quantifizieren. </li></ol>

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	<li>Lehr, H. A., Vollmar, B., Vajkoczy, P., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+fluorescence+microscopy+for+the+study+of+leukocyte+interaction+with+platelets+and+endothelial+cells.">Intravital fluorescence microscopy for the study of leukocyte interaction with platelets and endothelial cells.</a> Methods in enzymology. 300, 462-481 (1999).</li><li>Atherton, A., Born, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+investigations+of+the+adhesiveness+of+circulating+polymorphonuclear+leucocytes+to+blood+vessel+walls.">Quantitative investigations of the adhesiveness of circulating polymorphonuclear leucocytes to blood vessel walls.</a> The Journal of physiology. 222, 447-474 (1972).</li><li>Duerschmied, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+stimulates+platelet+receptor+shedding+by+tumor+necrosis+factor-alpha-converting+enzyme+(ADAM17).">Serotonin stimulates platelet receptor shedding by tumor necrosis factor-alpha-converting enzyme (ADAM17).</a> Journal of thrombosis and haemostasis : JTH. 7, 1163-1171 (2009).</li><li>Chauhan, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ADAMTS13:+a+new+link+between+thrombosis+and+inflammation.">ADAMTS13: a new link between thrombosis and inflammation.</a> The Journal of experimental medicine. 205, 2065-2074 (1084).</li><li>Baez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+open+cremaster+muscle+preparation+for+the+study+of+blood+vessels+by+in+vivo+microscopy.">An open cremaster muscle preparation for the study of blood vessels by in vivo microscopy.</a> Microvascular research. 5, 384-394 (1973).</li><li>Leister, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+peritoneal+cavity+chamber+for+intravital+microscopy+of+the+liver+under+conditions+of+pneumoperitoneum.">A peritoneal cavity chamber for intravital microscopy of the liver under conditions of pneumoperitoneum.</a> Surgical endoscopy. 17, 939-942 (2003).</li><li>Duerschmied, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+serotonin+promotes+the+recruitment+of+neutrophils+to+sites+of+acute+inflammation+in+mice.">Platelet serotonin promotes the recruitment of neutrophils to sites of acute inflammation in mice.</a> Blood. 121, 1008-1015 (2013).</li><li>Herr, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+fluoxetine+treatment+induces+slow+rolling+of+leukocytes+on+endothelium+in+mice.">Acute fluoxetine treatment induces slow rolling of leukocytes on endothelium in mice.</a> PloS one. 9, e88316 (2014).</li><li>Yardeni, T., Eckhaus, M., Morris, H. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vessel+wall+and+its+interactions.">The vessel wall and its interactions.</a> Blood. 111, 5271-5281 (2008).</li><li>Gawaz, M., Fateh-Moghadam, S., Pilz, G., Gurland, H. 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The authors have nothing to disclose.

Hier beschreiben wir die Vorbereitung und Durchführung der Intravitalmikroskopie der Gekröse Venen von Mäusen in vivo. Diese Methode gibt uns die Möglichkeit, Leukozyten-Endothel und Thrombozyten-Endothel-Interaktionen 11 direkt im lebenden Organismus zu beobachten. Als einer der ersten Reaktion auf das Endothel Entzündung aktiviert wird und interagiert mit von Leukozyten und Blutplättchen, was zu rollen, Adhäsion und Trans 12. Aber auch Leukozyten mit T...

Der Zweck dieses Protokolls ist es, ein vereinfachtes Verfahren der Intravitalmikroskopie der Gekröse Adern in lebenden Mäusen für die direkte Beobachtung der Leukozyten-Endothel und Leukozyten-Thrombozyten-Wechselwirkungen unter entzündlichen Erkrankungen zu beschreiben. Intravitalmikroskopie wurde entwickelt, um Leukozyten-Endothel-Interaktionen in vivo unter entzündlichen Erkrankungen 1,2, das ist nicht trivial, aber wichtig für das Verständnis entzündlicher Leukozytenrekrutierung und Funktion zu untersuchen. Die Methode beschreiben wir in diesem Protokoll wurde auf der Grundlage zuvor veröffentlichten Publikationen entwickelt. Ähnlich Visualisierung Thrombozyten Einbau in eine wachsende Thrombus 3 und parallel zu dem, was bereits früher veröffentlicht 4 ist ein exteriorisierten Mesenterium Venendurchlichtmikroskopie untersucht. Es gibt verschiedene andere Modelle von intra-Vitalmikroskopie wie dem Cremastermuskel von Ratten 5 oder Leber der Maus und Ratten 6. Intravitalmikroskopie Mesenterium Vene wurde entwickelt und in früheren Studien durch verschiedene Gruppen angewandt. Wir haben diese Technik verwendet werden, um Unterschiede in der Rekrutierung von Leukozyten zwischen Wildtyp-Mäusen und Tryptophan hydroxylase1 (Tph1) beobachten - / - Mäusen, die einen Mangel an nicht-neuronalen Serotonin und verglichen die Ergebnisse an Mäusen, deren Blutplättchen-Serotonin wurde pharmakologisch durch langfristige aufgebraucht sind Anwendung der ein selektiver Serotonin-Wiederaufnahmehemmer Fluoxetin 7. Wir haben auch Leukozyten-Endothel-Interaktionen nach akutem Fluoxetin-Behandlung 8 untersucht. In diesem Protokoll konzentrieren wir uns auf Intravitalmikroskopie Mesenterium Venen, weil dieses Modell kann schnell durchgeführt werden und ermöglicht gültigen Messungen der Leukozyten-Endothel und Blutplättchen-Leukozyten-Wechselwirkungen. Dies ist viel schwieriger und zeitraub in Intravitalmikroskopie an anderen Organen, wie etwa Knochen, Leber, Haut oder M. cremaster. Der Modushier beschriebene l ist nach herausforderndes mit einem entzündlichen Stimulus, wie beispielsweise eine intraperitoneale Injektion von Lipopolysaccharid ideal zur reproduzierbaren Beurteilung von entzündlichen Zell-Zell-Interaktion.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Rhodamine 6G</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;989-38-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharides from Escherichia coli 055:B5</td>
			<td>Sigma-Aldrich</td>
			<td>L2637&#160;</td>
			<td></td>
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intravital microscopy of leukocyte-endothelial and platelet-leukocyte interactions in mesenterial veins in mice

Intravitalmikroskopie ist eine Methode, die verwendet werden können, um verschiedene Prozesse in verschiedenen Regionen und Gefäße in lebenden Tieren zu untersuchen. In diesem Protokoll beschreiben wir Intravitalmikroskopie der Gekröse Adern. Dies kann in einem kurzen Zeitraum mit reproduzierbaren Ergebnissen, die Leukozyten-Endothel-Interaktionen in vivo durchgeführt werden. Wir beschreiben eine entzündliche Einstellung nach der LPS-Exposition des Endothels. Aber in diesem Modell kann man viele verschiedene Arten von entzündlichen Erkrankungen wie Bakterien, chemische oder biologische gelten, und untersuchen die Verabreichung von Arzneimitteln und deren direkte Auswirkungen auf die lebenden Tier und seine Auswirkungen auf die Rekrutierung von Leukozyten. Dieses Protokoll wurde erfolgreich in einer Reihe von verschiedenen Behandlungen von Mäusen und ihre Wirkungen auf entzündliche Reaktion in den Gefäßen eingesetzt. Hier beschreiben wir die Visualisierung von Leukozyten und Blutplättchen durch Fluoreszenzmarkierung diese mit Rhodamin 6G. Zusätzlich kann jede spezifische Bildgebung pe seinrformed durch gezielte fluoreszenzmarkierte Moleküle.

Ein Überblick über die in diesem Protokoll beschriebenen experimentellen Anordnung ist in Abbildung 1 dargestellt. Es zeigt eine Maus mit einem exteriorisierten Ileum-Loop und ihre Gefäße, die in Intravitalmikroskopie beobachtet werden kann, Fig. 2 zeigt bestimmte Aktivierungsgrad bei unbehandelten Tieren durch das Verfahren selbst. Aber es gibt fast keinen langsamen Rollen oder feste Adhäsion von Leukozyten im Vergleich zu LPS-behandelten Mäusen. 2 zeigt auch die...

Watch this Scientific Journal Video about Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice at JoVE.com

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

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.

Intravitalmikroskopie der Leukozyten-Endothel und Plättchen-Leukozyten-Wechselwirkungen in Mesenterialvenen 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 ...

intravital-microscopy-leukocyte-endothelial-platelet-leukocyte

Research

JoVE Journal

Immunology and Infection

12.0K Aufrufe.  Heart Center, University of Freiburg. 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.

Serie: Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

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.

Intravitalmikroskopie der Leistenbruch 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 ...

Forschung

Immunologie und Infektion

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.

Tracking-Neutrophil Intraluminale Crawling, transendotheliale Migration und Chemotaxis in Tissue durch Intravital Video-Mikroskopie

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.

Intravital Imaging of the Mouse Thymus mittels 2-Photonen-Mikroskopie

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.

Intravitalmikroskopie der Milz: Quantitative Analyse von Parasite Mobility und die Durchblutung

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

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.

Hochaufgelöste Intravitalmikroskopie Striped-Beleuchtung von Keimzentren

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

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.

Langzeitintravital Multiphotonen Mikroskopie Imaging von Immunzellen in gesunden und kranken Leber Mit CXCR6.Gfp Reporter Mäuse

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

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.

Abbilden Neutrophilen und Monozyten in Mesenterialvenen von Intravitalmikroskopie an narkotisierten Ratten in Echtzeit

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

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.

Vorbereitung der murinen mandibulären Speicheldrüse aufrecht Intravitalen Mikroskopie

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.

Bildgebung Zelle Interaktion in Trachealen Schleimhaut während der Influenza-Virus-Infektion mit Intravitalen zwei-Photonen-Mikroskopie

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. ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

Menschliche Colonoid Monolagen Wechselwirkungen zwischen Erreger, Kommensalen und Host Darmepithel zu studieren

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...