Nous remercions R. Heintzmann pour des discussions fructueuses lors de l&#39;élaboration de l&#39;approche-éclairage rayé, K. et A. Rajewsky Haberman pour fournir des souris transgéniques C57BL / 6 B1-8 GFP. Nous reconnaissons la Deutsche Forschungsgemeinschaft sous subvention NI1167/3-1 (RN), HA5354/4-1 et SFB633, A15 du projet (à AEH), la Charité sous-subvention Rahel Hirsch bourse (RN) pour le soutien financier. Nous reconnaissons en particulier le réseau JIMI pour des discussions fructueuses et soutien aux infrastructures

<ol>
	<li>Denk, W., Strickler, J. H., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+fluorescence+microscopy.">Two-photon laser scanning fluorescence microscopy.</a> Science. 248 (4951), 73-76 (1990).</li><li>Siffrin, V., 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=In+vivo+imaging+of+partially+reversible+th17+cell-induced+neuronal+dysfunction+in+the+course+of+encephalomyelitis.">In vivo imaging of partially reversible th17 cell-induced neuronal dysfunction in the course of encephalomyelitis.</a> Immunity. 33 (3), 424-436 (2010).</li><li>Esplugues, E., 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=Control+of+Th17+cells+occurs+in+the+small+intestine.">Control of Th17 cells occurs in the small intestine.</a> Nature. 475 (7357), 514-518 (2011).</li><li>Hauser, A. E., 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=Definition+of+germinal-center+B+cell+migration+in+vivo+reveals+predominant+intrazonal+circulation+patterns.">Definition of germinal-center B cell migration in vivo reveals predominant intrazonal circulation patterns.</a> Immunity. 26 (5), 655-667 (2007).</li><li>Cahalan, M. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choreography+of+cell+motility+and+interaction+dynamics+imaged+by+two-photon+microscopy+in+lymphoid+organs.">Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs.</a> Annu. Rev. Immunol. 26, 585-626 (2008).</li><li>Herz, J., 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=Expanding+two-photon+intravital+microscopy+to+the+infrared+by+means+of+optical+parametric+oscillator.">Expanding two-photon intravital microscopy to the infrared by means of optical parametric oscillator.</a> Biophys. J. 98 (4), 715-723 (2010).</li><li>Hell, S. W., Rittweger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopy:+Light+from+the+dark.">Microscopy: Light from the dark.</a> Nature. 461 (7267), 1069-1070 (2009).</li><li>Ding, J. B., Takasaki, K. T., Sabatini, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supraresolution+imaging+in+brain+slices+using+stimulated-emission+depletion+two-photon+laser+scanning+microscopy.">Supraresolution imaging in brain slices using stimulated-emission depletion two-photon laser scanning microscopy.</a> Neuron. 63 (4), 429-437 (2009).</li><li>Bianchini, P., 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=Single-wavelength+two-photon+excitation+-+stimulated+emission+depletion+(SW2PE-STED)+super-resolution+imaging.">Single-wavelength two-photon excitation - stimulated emission depletion (SW2PE-STED) super-resolution imaging.</a> Proc. Natl. Acad. Sci. U.S.A. 109 (17), 6390-6393 (2012).</li><li>Berning, S., 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=Nanoscopy+in+a+living+mouse+brain.">Nanoscopy in a living mouse brain.</a> Science. 335 (6068), 551 (2012).</li><li>York, A. G., 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=Resolution+doubling+in+live,+multicellular+organisms+via+multifocal+structured+illumination+microscopy.">Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy.</a> Nat. Methods. 9, 749-754 (2012).</li><li>Gu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advanced optical imaging. , (2001).</li><li>Cha, J. W., Ballesta, J., So, P. T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shack-Hartmann-wave+front-sensor-based+adaptive+optics+system+for+multi-photon+microscopy.">Shack-Hartmann-wave front-sensor-based adaptive optics system for multi-photon microscopy.</a> J. Biomed. Opt. 15 (4), (2010).</li><li>Booth, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+optics+in+microscopy.">Adaptive optics in microscopy.</a> Phil. Trans. R. Soc. A. 365, 2829-2843 (2007).</li><li>Niesner, R., 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=The+power+of+single+and+multibeam+two-photon+microscopy+for+high-resolution+and+high-speed+deep+tissue+and+intravital+imaging.">The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging.</a> Biophys. J. 93 (7), 2519-2529 (2007).</li><li>R&#252;ckel, M., Mack-Bucher, J. A., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+wave-front+correction+in+TPM+using+coherence-gated+wave-front+sensing.">Adaptive wave-front correction in TPM using coherence-gated wave-front sensing.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 17137-17142 (2006).</li><li>Tang, J., Germain, R. N., Cui, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superpenetration+optical+microscopy+by+iterative+multiphoton+adaptive+compensation+technique.">Superpenetration optical microscopy by iterative multiphoton adaptive compensation technique.</a> Proc. Natl. Acad. Sci. USA. 109 (22), 8434-8439 (2012).</li><li>Ji, N., Milkie, D. E., Betzig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+optics+via+pupil+segmentation+for+high+resolution+imaging+in+biological+tissues.">Adaptive optics via pupil segmentation for high resolution imaging in biological tissues.</a> Nat. Methods. 7, 141-147 (2009).</li><li>Lu, J., 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=Super-resolution+laser+scanning+microscopy+through+spatiotemporal+modulation.">Super-resolution laser scanning microscopy through spatiotemporal modulation.</a> Nano Lett. 9 (11), 3883-3889 (2009).</li><li>Andresen, V., 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=High-resolution+intravital+microscopy.">High-resolution intravital microscopy.</a> PLoS ONE. 7 (12), (2012).</li><li>Debarre, 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=Adaptive+optics+for+structured-illumination.">Adaptive optics for structured-illumination.</a> Opt. Exp. 16 (13), 9290-9305 (2008).</li></ol>

Volker Andresen est un actionnaire de LaVision Biotec GmbH, Bielefeld, Allemagne. Les autres auteurs n&#39;ont pas de conflits d&#39;intérêts.

Le but de l&#39;imagerie optique intravitale est de visualiser dynamiquement et fonctionnellement la motilité cellulaire et interactions pour comprendre le tissu et la fonction des organes en santé et la maladie 5. La technologie la plus puissante pour ce faire, multi-photons laser microscopie, doit encore surmonter les limitations liées à la vague distorsions avant, la diffusion, l&#39;acquisition lente, photoblanchiment et le photovieillissement, qui limitent la résolution spatiale, le contraste ainsi ...

Deux photons laser microscopie (TPLSM), avec ses avantages pour l&#39;imagerie des tissus profonds liés à infrarouge ultra-courte excitation pulsée 1, a révolutionné notre point de vue sur les processus vitaux sur un seul niveau cellulaire en révélant la motilité et l&#39;interaction de divers modèles sous-ensembles de cellules dans des animaux vivants 5.2. Cependant, la technologie actuelle est encore insuffisant pour élucider les mécanismes de fonctionnement des organes et le dysfonctionnement comme une condition préalable pour le développement de nouvelles stratégies thérapeutiques, car il ne rend rares informations sur les bases moléculaires de la réponse cellulaire dans les tissus de la santé et de la maladie. La technologie actuelle permet seulement une fenêtre spatiale de quelques centaines de microns en raison de la dispersion des vagues et des effets de distorsion de front sur ​​la résolution spatiale et rapport signal à bruit de 6, ce qui est particulièrement évident dans le tissu très compact d&#39;animaux adultes. Ces vagues de diffusion et des effets de distorsion avant sont dues à une une très hétérogènesd la distribution anisotrope de l&#39;indice de réfraction dans le tissu, ce qui conduit dans une première étape à une dégradation de la résolution spatiale dépend de la profondeur en 3D, et enfin à la perte totale du signal provenant de photons d&#39;excitation balistiques, à savoir la perte de rapport signal à bruit. En termes de bioscientifiques et les applications biomédicales des tissus profonds, cela signifie que la technologie actuelle n&#39;est pas en mesure de révéler sans équivoque communication cellulaire parce que la mauvaise résolution entraîner des interactions faussement positifs alors que la diminution de rapport signal-sur-bruit causerait le système de négliger certains interactions entre les structures sombres. Afin de détecter sans équivoque interactions cellulaires de façon dynamique, une résolution spatiale très améliorée est nécessaire profond dans le tissu. Les techniques de nanoscopie puissants actuellement introduites sur la base d&#39;algorithmes numériques spéciaux, par exemple des approches structure éclairage, sur l&#39;épuisement du premier état ​​excité, par exemple </em&gt; STED, RESOLFT, ou sur la localisation molécule, par exemple dSTORM, PALM, ont trouvé de nombreuses applications dans les cellules fixes ainsi que dans des cultures de cellules vivantes 7. Toutefois, afin d&#39;étendre ces applications à des coupes de tissus, des tissus vivants et organismes que nous avons encore besoin de surmonter les difficultés techniques graves. Excitation à deux photons STED avec différentes longueurs d&#39;onde, ainsi que d&#39;une seule longueur d&#39;onde (sw2PE-STED) pour l&#39;excitation et l&#39;émission stimulée a été appliquée pour améliorer la résolution latérale dans des tranches de cerveau 8 ou dans des matrices artificielles à cellules incluses 9, respectivement, dans le même résolution axiale comme TPLSM standard. Utilisation d&#39;un photon STED, la dynamique des épines dendritiques peuvent être imagées à la surface du cortex cérébral (jusqu&#39;à 10 à 15 um de profondeur) dans une souris vivante Thy1 EGFP à une résolution de 67 nm 10. Un outil polyvalent pour la biologie du développement est fourni par la microscopie structuré-éclairage multifocale, qui fournit deux fois améliorée 2Drésolution. Cependant, cette technique peut être utilisée que dans les organismes avec une faible propension de diffusion de la lumière tels que des embryons de poisson zèbre 11. Cependant, aucune de ces techniques peut être appliquée dans le tissu très diffusant des animaux adultes à plusieurs centaines de micromètres, qui sont cruciales pour les modèles de la recherche biomédicale et clinique des maladies avec la survenue après la naissance. Indépendant de l&#39;approximation utilisée pour calculer la forme de l&#39;avant onde limitée par la diffraction, c&#39;est à dire la fonction d&#39;étalement de point (PSF), après focalisation par une lentille, la largeur de la PSF long de l&#39;axe optique (résolution axiale) est au moins trois fois plus grande que la largeur de la PSF perpendiculaire à l&#39;axe optique (résolution latérale) 12. Déformations de l&#39;onde avant d&#39;ordres différents quantifiés par les coefficients de Zernike modifier considérablement la forme du front d&#39;onde de l&#39;onde électromagnétique concentré dans l&#39;imagerie des tissus profonds menant à beaucoup plus PSF, en particulier le long de la fibreal axe 13-15. Par conséquent, à la fois les lois de la diffraction et les effets de distorsion de front d&#39;onde point à la résolution le long de l&#39;axe optique que le facteur limitant dans l&#39;imagerie des tissus profonds. Considérant que des techniques nanoscopie concentrent sur la lutte contre les limites de la diffraction seulement, une technologie qui améliore la résolution axiale et le contraste en compensant à la fois la diffraction et les effets de distorsion de front d&#39;onde est nécessaire pour intravitale imagerie à haute résolution. Idéalement, cette technique devrait également être suffisamment rapide pour permettre un suivi de la dynamique cellulaire. La correction en temps réel des aberrations PSF et la perte de contraste à l&#39;aide de l&#39;optique adaptative dans TPLSM a été largement étudié et amélioré dans la dernière décennie 13,14,16-18 et il est donc le meilleur choix actuellement disponibles conduisant à une meilleure gestion de l&#39;excitation balistique photons 14. Cependant, en raison du fait que la plupart des vagues avant correction approches utilisées en optique adaptative sont itérative et qu&#39;ils have être répété pour les petites surfaces (quelques 10 x 10 um 2) en raison de la forte hétérogénéité de l&#39;indice de réfraction dans le tissu, la vitesse d&#39;acquisition est significativement plus faible que nécessaire pour la motilité cellulaire et la communication d&#39;image. En outre, la limite physique en optique adaptative améliorée TPLSM est toujours déterminée par diffraction. Modulation spatiale de l&#39;illumination (SPIN) et modulation temporelle sur le côté de détection (SPADE) ont été théoriquement proposé à appliquer à la microscopie à balayage laser pour améliorer la résolution. Leur application pratique dans l&#39;imagerie intravitale reste encore à être démontrée 19. Dans l&#39;ensemble, il ya une forte demande pour le développement de technologies qui améliorent la résolution pour l&#39;imagerie des tissus profonds chez les animaux adultes vivant. Dans ce travail, nous obtenons la modulation spatiale du motif d&#39;excitation en commandant le processus de balayage à faisceaux multiples rayé-illumination multi-photons laser-smicroscopie de la conserve (Mo-SI-MPLSM) 20. Contrairement aux approches d&#39;éclairage structuré, dans lequel la section d&#39;excitation transversale du faisceau est modulé spatialement, on utilise seulement le processus de balayage pour réaliser la modulation spatiale de l&#39;excitation. En élargissant l&#39;excitation à une longueur d&#39;onde plus, nous sommes en mesure d&#39;améliorer à la fois la résolution spatiale et le rapport signal-sur-bruit dans les tissus profonds de haute diffusion tissulaire (par exemple des ganglions lymphatiques, chemin libre diffusion 47 um 6), indépendamment de l&#39;optique signal non-linéaire nous détectons, par exemple fluorescence, génération de second harmonique ou autre fréquence mélange phénomène. En utilisant cette approche à des longueurs d&#39;onde d&#39;excitation à 900 nm, nous sommes en mesure de façon dynamique l&#39;image des structures de protéines cellulaires à l&#39;échelle de quelques molécules dans les centres germinaux des ganglions lymphatiques de souris. Ainsi, on peut mieux visualiser l&#39;interaction entre les unités de support d&#39;antigène sur la surface des cellules dendritiques folliculaires et les cellules B dans le processus de palpage contre lagen au cours de la réponse immunitaire dans les organes lymphoïdes secondaires.

<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

Suivi communication cellulaire par intravitale profond des tissus multi-microscopie biphotonique est la clé pour comprendre le destin des cellules immunitaires dans les échantillons de tissus et d&#39;organes d&#39;épaisseur dans la santé et la maladie. En contrôlant le motif de balayage dans la microscopie multi-photons et en appliquant des algorithmes numériques appropriés, nous avons développé une approche rayé-illumination, ce qui nous a permis d&#39;atteindre trois fois meilleure résolution axiale et une amélioration de rapport signal sur bruit, c&#39;est à dire l&#39;inverse, en plus de profondeur de 100 um de tissu dans le tissu des organes lymphoïdes haute diffusion par rapport à la microscopie multi-photons standard. La vitesse d&#39;acquisition ainsi que photoblanchiment et le photovieillissement effets sont similaires à la technique à base de photo-multiplicateur-standard, tandis que la profondeur d&#39;imagerie a été légèrement inférieure en raison de l&#39;utilisation de détecteurs de champ. En utilisant l&#39;approche-éclairage rayé, nous sommes en mesure d&#39;observer la dynamique des dépôts de complexes immuns sur les cellules dendritiques folliculaires secondaires ̵1; au niveau de quelques molécules de protéines dans les centres germinatifs.

La résolution spatiale dans les ganglions lymphatiques Les dimensions de la fonction d&#39;étalement de point efficace (EPSF) correspondent à la résolution spatiale d&#39;un microscope 12. Nous avons mesuré la fonction tridimensionnelle en acquérant le signal de deuxième génération d&#39;harmoniques de fibres de collagène dans les ganglions lymphatiques par notre MB-SI-TPLSM par rapport à l&#39;établis des techniques de microscopie à balayage laser à deux photons, c...

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

Haute résolution intravitale imagerie avec un contraste accru jusqu&#39;à 120 um de profondeur dans les ganglions lymphatiques de la souris adulte est obtenue en modulant spatialement le modèle d&#39;excitation d&#39;un multi-focal microscope à deux photons. En 100 profondeur pm nous avons mesuré des résolutions de 487 nm (latéraux) et 551 nm (axial), contournant ainsi les limites de diffusion et de diffraction.

Très Résolu microscopie intravitale Striped-éclairage de centres germinaux

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 vues.  Leibniz Institute.  Haute résolution intravitale imagerie avec un contraste accru jusqu'à 120 um de profondeur dans les ganglions lymphatiques de la souris adulte est obtenue en modulant spatialement le modèle d'excitation d'un multi-focal microscope à deux photons. En 100 profondeur pm nous avons mesuré des résolutions de 487 nm (latéraux) et 551 nm (axial), contournant ainsi les limites de diffusion et de diffraction. 

Vidéo: 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.

Microscopie intravitale du ganglion lymphatique inguinal

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 ...

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Immunologie et infection

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.

Suivi des rampants neutrophiles intraluminale, migration trans et le chimiotactisme dans les tissus par microscopie intravitale Vidéo

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.

Imagerie intravitale du Thymus souris utilisant la 2-microscopie biphotonique

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.

Microscopie intravitale de la rate: Analyse quantitative de la mobilité des parasites et la circulation sanguine

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.

Microscopie intravitale imagerie du foie suivante<em&gt; Leishmania</em&gt; Infection: Une évaluation de l&#39;hémodynamique hépatiques

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.

Interactions Microscopie intravitale des leucocytes endothéliaux et plaquettes-leucocytes dans mésentériques veines chez les souris

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.

Imagerie neutrophiles et des monocytes dans les veines mésentérique par Microscopie intravitale sur des souris anesthésiées en temps réel

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.

Swab Méthode d&#39;échantillonnage pour la détection des norovirus humains sur les surfaces

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.

Préparation du murin des glandes salivaires sous-maxillaires pour la microscopie intravitale verticale

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.

Imagerie cellulaire Interaction dans la muqueuse trachéale au cours de l’infection par le Virus grippal à l’aide de la microscopie intravitale biphotonique

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. ...