This project was supported by grants NIH NIGMS 8P41 GM103540-28 and P50-GM076516

Préparation de l&#39;échantillon 1 <ol><li> Maintenir des cellules CHOK1 dans des flacons de culture de tissus à l&#39;aide de DMEM supplémenté avec 10% de sérum bovin foetal et 100 UI / ml de pénicilline 50 pg / ml de streptomycine. Incuber les cellules dans un incubateur humidifié à 5% de CO2 à 37 ° C. </li><li> Récolte et ensuite sur l&#39;assiette cellules CHOK1 un diamètre de micro-puits de 14 mm avec une épaisseur de 0,16 mm de surface. cellules de semences pour la densité optimale pour l&#39;imagerie, environ 60-70% de confluence. </li><li> Incuber les cellules pendant une nuit à 37 ° C, 5% CO 2. Laver les cellules trois fois dans du HBSS (solution saline tamponnée de Hank), et incuber les cellules dans une solution contenant 50 nM de Lysotracker DND26 vert et 150 nM de la tubuline traqueur vert. Incuber les cellules pendant 1 heure à 37 ° C. Etape facultative: Avant l&#39;incubation, effectuer une tache supplémentaire avec un colorant matrice de coloration mitochondriale (25 nM). </li><li> Laver les cellules trois fois dans du HBSS pour éliminer tout colorant non lié. Ajouter milieu de croissance DMEM frais avant imagtion des cellules. </li></ol> 2. Configurations microscope Le microscope pour le suivi des particules décrites dans le protocole vidéo est montée sur le châssis d&#39;un microscope inversé disponible dans le commerce (Figure 4). Cependant modules commerciaux pour le suivi des particules orbitale 3D sont maintenant disponibles. <ol><li> Utilisez un II cohérente Chameleon Ultra-Ti: source de lumière d&#39;excitation laser femtoseconde Sa, avec une gamme accordable sortie d&#39;onde comprise entre 690 nm-1040 nm. </li><li> Assurez-vous que le faisceau laser soit aligné sur l&#39;orifice arrière du microscope en utilisant un miroir à revêtement IR. Le faisceau laser est atténué à l&#39;aide d&#39;une lame demi-onde tournante suivie d&#39;un polariseur linéaire de calcite. Atténuer le faisceau de telle sorte que la puissance moyenne de l&#39;échantillon est compris entre 0,5 et 2 mW. NOTE: Le faisceau laser est réfléchi par une paire de miroirs actionnés galvanomètre moteurs qui permettent le contrôle de la position et de la trajectoire du faisceau focalisé dans le plan de l&#39;échantillon. Dans unconfiguration typique du faisceau laser collimaté sortant de miroirs de galvanomètre agrandi (10X) passant à travers un élargisseur de faisceau, avant d&#39;entrer à l&#39;arrière de l&#39;objectif de microscope, après réflexion sur courte passe-miroir dichroïque. </li><li> Collecter la lumière de fluorescence provenant de l&#39;échantillon en plaçant un objectif de l&#39;eau à grande ouverture numérique (60X, NA 1,2) dans le trajet de la lumière. Choisir un cube de filtre de fluorescence en fonction de la longueur d&#39;onde d&#39;émission souhaitées dans une ou l&#39;autre configuration d&#39;un ou deux canaux. Utiliser des filtres passe-bande d&#39;émission pour sélectionner en outre la gamme spectrale de la fluorescence émise. </li><li> Placer l&#39;échantillon sur une platine motorisée, et ajuster l&#39;amende mouvement de l&#39;objectif de microscope à l&#39;aide d&#39;un piézo-contrôleur objectif. </li><li> Diriger la lumière à partir du cube de filtre dans des tubes photomultiplicateurs où le signal est discriminé et envoyé à une carte E / S d&#39;acquisition de données numériques. REMARQUE: Ordinateur généré des formes d&#39;onde sont la sortie analogique de la carte E / S et sont provided pour les scanners de contrôle électronique. L&#39;entrée de comptage de photons à partir des photomultiplicateurs et le signal de sortie pour les scanners sont mesurés et contrôlés par l&#39;intermédiaire de la carte d&#39;E / S par le Laboratoire de logiciel de dynamique de fluorescence SimFCS. </li></ol> 3. Imaging <ol><li> Placez les cellules sur la scène et de se concentrer en utilisant un éclairage lumière transmise. </li><li> Mettre à balayage de trame imagerie pour identifier les cellules qui ont incorporé le colorant et de visualiser les microtubules sous-jacents. Identifier la zone initiale où le mouvement des vésicules est présent. </li><li> Une fois une vésicule isolé est identifié, définissez les paramètres suivants pour le suivi orbital: <ol><li> Sélectionner le rayon de l&#39;orbite de définir la taille du balayage circulaire en fonction de la taille de la particule est suivie. Pour un émetteur ponctuel, définir le rayon de l&#39;orbite égale à la taille de la fonction de flou (PSF) du faisceau d&#39;excitation pour maximiser la sensibilité et la réponse. </li><li> Réglez le axialdistance pour définir la distance entre les deux orbites qui sont exécutées pour localiser la position de la particule suivant la direction axiale. Réglez la distance axiale à 1,5-3 fois la taille de la PSF. </li><li> Définir la durée de séjour en fonction de la luminosité de la particule pour définir le temps consacré à chaque point de l&#39;orbite, ce qui permettra également de déterminer le taux de photo-blanchiment. Utilisez un temps d&#39;arrêt entre 10-100 microsecondes. </li><li> Définissez le nombre de points dans chaque orbite à 64 ou 128 pour obtenir des périodes orbitales de l&#39;ordre de 4-32 ms et fournir une haute résolution temporelle pour déterminer la position de la particule. </li></ol></li><li> Changer le signal de décalage à courant continu envoyée à miroirs afin de centrer le faisceau sur la particule par l&#39;intermédiaire de l&#39;interface utilisateur graphique par l&#39;intermédiaire d&#39;un curseur de sélection dans l&#39;image de balayage de trame. </li><li> Commencer à suivre en changeant de mode de raster-imagerie microscope à balayage orbital. </li><li> Activer la collecte de commentaires et données. </li></ol> 4. TrajectAnalyse ory <ol><li> Utilisez le logiciel pour afficher la fluorescence collectées à chaque point le long de l&#39;orbite et à chaque point de temps sous la forme d&#39;un «tapis d&#39;intensité». L&#39;intensité recueillie sur le tapis contient des informations sur l&#39;interaction de la particule avec d&#39;autres objets lumineux. Utiliser le logiciel pour afficher à la fois les informations de trajectoire (c&#39;est à dire le déplacement continu au cours du temps de la x, scanners y et des z -piezo) ainsi que l&#39;intensité de fluorescence collecté par le tube photomultiplicateur au cours du temps sous forme de séries chronologiques. </li><li> Utiliser la représentation de série de temps et l&#39;information &quot;d&#39;intensité de tapis» à sélectionner uniquement une région d&#39;intérêt dans la trajectoire. </li><li> Utiliser le logiciel pour afficher une projection 2D de la partie sélectionnée de la trajectoire des particules. Sélectionnez les contrôles appropriés à code couleur de la trajectoire selon l&#39;intensité de fluorescence des particules. </li><li> Sélectionnez l&#39;option pour afficher la pagearticle trajectoire en 3D, et un code couleur en fonction de l&#39;intensité de fluorescence. Tournez la trajectoire en 3D en utilisant les contrôles de visualiser les caractéristiques de la motion de lysosome le long des microtubules. </li></ol>

<ol>
	<li>So, P. T., Dong, C. Y., Masters, B. R., Berland, K. M. <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+excitation+fluorescence+microscopy.">Two photon excitation fluorescence microscopy.</a> Annu Rev Biomed Eng. 2, 399-429 (2000).</li><li>Dupont, A., Lamb, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+three+dimensional+single+particle+tracking.">Nanoscale three dimensional single particle tracking.</a> Nanoscale. 3, 4532-4541 (2011).</li><li>Estrada, L. C., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+nanometer+images+of+biological+fibers+by+directed+motion+of+gold+nanoparticles.">3D nanometer images of biological fibers by directed motion of gold nanoparticles.</a> Nano Lett. 11, 4656-4660 (2011).</li><li>Kis-Petikova, K., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distance+measurement+by+circular+scanning+of+the+excitation+beam+in+the+two+photon+microscope.">Distance measurement by circular scanning of the excitation beam in the two photon microscope.</a> Microscopy Research and Technique. 63, 34-49 (2004).</li><li>Levi, V., Ruan, Q., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3+D+particle+tracking+in+a+two+photon+microscope+application+to+the+study+of+molecular+dynamics+in+cells.">3 D particle tracking in a two photon microscope application to the study of molecular dynamics in cells.</a> Biophys J. 88, 2919-2928 (2005).</li><li>Lund, F. W., Wustner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+single+particle+tracking+and+temporal+image+correlation+spectroscopy+for+quantitative+analysis+of+endosome+motility.">A comparison of single particle tracking and temporal image correlation spectroscopy for quantitative analysis of endosome motility.</a> Journal of Microscopy. 252, 169-188 (2013).</li><li>Nath, 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=Spreading+of+Neurodegenerative+Pathology+via+Neuron+to+Neuron+Transmission+of+beta+Amyloid.">Spreading of Neurodegenerative Pathology via Neuron to Neuron Transmission of beta Amyloid.</a> Journal of Neuroscience. 32, 8767-8777 (2012).</li><li>Balint, S., Vilanova, I. V., Alvarez, A. S., Lakadamyali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlative+live+cell+and+superresolution+microscopy+reveals+cargo+transport+dynamics+at+microtubule+intersections.">Correlative live cell and superresolution microscopy reveals cargo transport dynamics at microtubule intersections.</a> Proceedings of the National Academy of Sciences of the United States of America. 110, 3375-3380 (2013).</li><li>Huotari, J., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endosome+maturation.">Endosome maturation.</a> Embo Journal. 30, 3481-3500 (2011).</li><li>Ragan, T., So, P. T., Kwon, H. S., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+particle+tracking+on+the+two+photon+microscope.">3D particle tracking on the two photon microscope.</a> Multiphoton Microscopy in the Biomedical Sciences. 2, 247-258 (2001).</li><li>Konig, K., So, P. T. C., Mantulin, W. W., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+response+to+near-infrared+femtosecond+laser+pulses+in+two+photon+microscopes.">Cellular response to near-infrared femtosecond laser pulses in two photon microscopes.</a> Optics Letters. 22, 135-136 (1997).</li><li>Caviston, J. P., Holzbaur, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+motors+at+the+intersection+of+trafficking+and+transport.">Microtubule motors at the intersection of trafficking and transport.</a> Trends Cell Biol. 16, 530-537 (2006).</li><li>Cardarelli, F., Lanzano, L., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+directed+molecular+motion+in+the+nuclear+pore+complex+of+live+cells.">Capturing directed molecular motion in the nuclear pore complex of live cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, 9863-9868 (2012).</li></ol>

The authors have nothing to disclose.

Malgré les énormes progrès des techniques de fluorescence en microscopie et instrumentations au cours des dernières années, la réalisation de trajectoires de particules fluorescentes en trois dimensions avec une résolution temporelle est restée un défi dans le domaine. Si une haute résolution temporelle a été atteinte particules de repérage à deux dimensions, l&#39;extension à la direction axiale apporte généralement une réduction drastique de la réponse en fréquence du système 10. <p...

Un grand nombre d&#39;approches ont été développées à ce jour pour suivre les particules fluorescentes dans trois dimensions à l&#39;aide d&#39;un microscope. La plupart des approches reposent sur l&#39;utilisation de caméras rapides, parfaitement adapté pour suivre en deux dimensions, typiquement combinées avec les adaptations personnalisées de l&#39;optique d&#39;émission du microscope pour réaliser un suivi dans le sens axial. microscopes à balayage laser (soit confocale ou deux photons) conventionnelle peut suivre une particule fluorescente en effectuant une séquence temporelle de z-piles, même si ce processus est généralement beaucoup de temps, et donne la résolution de temps raisonnable (10 Hz) que si la particule étant suivi est conservé dans le centre d&#39;une petite région de formation d&#39;image tramée par un mécanisme de rétroaction actif 2. L&#39;idée de l&#39;immobilisation de la particule est la base de la méthode de suivi orbital. Au lieu d&#39;une trame numériser une orbite circulaire est réalisée autour de la particule fluorescente. L&#39;intensité de la fluorescence le long del&#39;orbite localise précisément la position des particules 4. La position localisée de la particule peut ensuite être utilisé pour actionner les scanners de microscope et recentrer les orbites de la position de la particule. Les scanners galvanométriques du microscope sont entraînés par une tension analogique. La composante alternative de cette tension permet d&#39;effectuer une orbite avec le faisceau laser focalisé, c&#39;est à dire un sinus et une onde cosinus appliquée à des miroirs X et Y balayage permettant d&#39;effectuer une orbite circulaire. Le décalage en courant continu du signal facilite le changement de la position du centre de l&#39;orbite. Une fois la période de l&#39;orbite est déterminée et la forme d&#39;onde pour le signal en courant alternatif est configuré, le système de rétroaction a besoin pour mettre à jour uniquement la composante continue du signal. Un logiciel capable de lire le signal fluorescent recueillies par les détecteurs est nécessaire pour contrôler les scanners et les détecteurs, de calculer la position de la particule et mettre à jour la DC horsdéfinir. Pour un retour réussi imagerie d&#39;un choix judicieux des paramètres orbitaux (taille et de synchronisation) est nécessaire et ces paramètres doivent être ajustés en fonction des caractéristiques des particules fluorescentes, il est nécessaire de suivre. Les fondements physiques et mathématiques de la technique ont été décrites dans le passé de 4,5 pour les applications 2D et 3D. Dans ce protocole, nous décrivons brièvement les principaux composants matériels de l&#39;installation, et plus en détail le choix des paramètres et la préparation de l&#39;échantillon nécessaire pour une expérience typique qui nous a permis suite au déplacement d&#39;un lysosome à une haute résolution temporelle dans une cellule vivante.

<table border="0" cellpadding="0" cellspacing="0" width="525">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:197px;">Name</td>
			<td style="width:160px;">Company</td>
			<td style="width:168px;">Model</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lysotracker DND 26</td>
			<td>Life technologies</td>
			<td>L-7526</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tubuline tracker Green</td>
			<td>Life technologies</td>
			<td>T34075</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mitotracker RED</td>
			<td>Life technologies</td>
			<td>M7512</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coherent Chamelon- ultra II TI</td>
			<td>Coherent</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcite polarize</td>
			<td>Melles Griot Glan&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Galvanometer-motor mirror</td>
			<td>Cambridge technologies</td>
			<td>M 6350</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dichroic mirror</td>
			<td>Chroma technologies</td>
			<td>700 DCSPXR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Motorized stage</td>
			<td>ASI</td>
			<td>MS2000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Piezo&#160;</td>
			<td>PI</td>
			<td>P721-LLQ</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Photomultiplier tube</td>
			<td>Hamamatsu</td>
			<td>H7422P-40</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Data acquisition card</td>
			<td>IO tech</td>
			<td>PCI 1128-4000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaging software</td>
			<td>LFD</td>
			<td>Global for images-SimFCS</td>
		</tr>
	</tbody>
</table>

3d orbital tracking in a modified two-photon microscope: an application to the tracking of intracellular vesicles

L&#39;objectif de ce protocole vidéo est de discuter de la façon d&#39;effectuer et d&#39;analyser une expérience fluorescente en trois dimensions suivi de particules orbital en utilisant un microscope à deux photons modifié 1. Contrairement aux approches classiques (de balayage de trame ou à grand champ basé sur une pile de cadres), le suivi orbitale 3D permet de localiser et de suivre avec une grande spatiale (10 précision nm) et la résolution temporelle (réponse en fréquence de 50 Hz) le déplacement 3D de une particule fluorescente déplacer sur la longueur des échelles de centaines de microns 2. La méthode est basée sur un algorithme de contre-réaction qui commande le matériel de deux photons microscope à balayage laser pour effectuer une orbite circulaire autour de l&#39;objet à suivre: le mécanisme de rétroaction maintient l&#39;objet fluorescent dans le centre de commande du déplacement de le faisceau de balayage 5.3. Pour démontrer les avantages de cette technique, nous avons suivi un organite se déplaçant rapidement, le lysosome, dans alcellule iving 6,7. Les cellules ont été étalées selon des protocoles standard, et colorées à l&#39;aide d&#39;un colorant dans le commerce lysosome. Nous discutons brièvement la configuration matérielle et plus en détail le logiciel de contrôle, à réaliser une expérience de suivi orbitale 3D à l&#39;intérieur des cellules vivantes. On discute en détail les paramètres nécessaires afin de contrôler le microscope à balayage et permettre le mouvement de la poutre sur une orbite fermée autour de la particule. Nous concluons en démontrant comment cette méthode peut être utilisée efficacement pour suivre le mouvement rapide d&#39;un lysosome marqué le long des microtubules en 3D dans une cellule vivante. Les lysosomes peuvent se déplacer à des vitesses de l&#39;ordre de 0,4-0,5 um / s, en affichant un mouvement généralement dirigé le long du réseau de microtubules 8.

Selon ce protocole suivi rapide de particules isolées 3D peut être effectué à l&#39;intérieur de cellules vivantes à l&#39;aide d&#39;un microscope à deux photons à jour pour suivre le déplacement des lysosomes marquées par fluorescence. L&#39;expérience réalisée consiste en un suivi de lysosome isolée se déplaçant à l&#39;intérieur de la cellule après le processus de maturation de l&#39;endosome 9. Les lysosomes ont été colorées en utilisant un colorant vert fluorescent et excités à 9...

Watch this Scientific Journal Video about 3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles at JoVE.com

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

Suivi 3D orbital dans un microscope à deux photons à jour: Une application au suivi des vésicules intracellulaires

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

3d-orbital-tracking-modified-two-photon-microscope-an-application-to

Research

JoVE Journal

Bioengineering

10.1K vues.  University of California, Irvine. In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope. 

Vidéo: 3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal hyperglycemia. As an alternative treatment option to exogenous insulin injection, transplantation of functional pancreatic tissue has been explored1,2. This approach offers the promise of a more natural, long-term restoration of normoglycemia. Protection of the donor tissue from the host's immune system is required to prevent rejection and encapsulation is a method used to help achieve this aim.
Biologically-derived materials, such as alginate3 and agarose4, have been the traditional choice for capsule construction but may induce inflammation or fibrotic overgrowth5 which can impede nutrient and oxygen transport. Alternatively, synthetic poly(ethylene glycol) (PEG)-based hydrogels are non-degrading, easily functionalized, available at high purity, have controllable pore size, and are extremely biocompatible,6,7,8. As an additional benefit, PEG hydrogels may be formed rapidly in a simple photo-crosslinking reaction that does not require application of non-physiological temperatures6,7. Such a procedure is described here. In the crosslinking reaction, UV degradation of the photoinitiator, 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959), produces free radicals which attack the vinyl carbon-carbon double bonds of dimethacrylated PEG (PEGDM) inducing crosslinking at the chain ends. Crosslinking can be achieved within 10 minutes. PEG hydrogels constructed in such a manner have been shown to favorably support cells7,9, and the low photoinitiator concentration and brief exposure to UV irradiation is not detrimental to viability and function of the encapsulated tissue10. While we methacrylate our PEG with the method described below, PEGDM can also be directly purchased from vendors such as Sigma.
An inherent consequence of encapsulation is isolation of the cells from a vascular network. Supply of nutrients, notably oxygen, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact &beta;-cells whose insulin secretory function is highly dependent on oxygen11-13. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated14. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling15. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously15. The virus is stored in 10% glycerol at -80&deg; C as many 150 &mu;L aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 1010 pfu/mL. 
Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion15. Western blot analysis of encapsulated cells in 20% and 1% oxygen also showed a significant increase in HIF-1&alpha; for cells cultured in the low oxygen conditions which correlates with the expression of the DsRed DR protein.

A method for photo-encapsulation of cells in a crosslinked PEG hydrogel is described. Hypoxic signaling within encapsulated murine insulinoma (MIN6) aggregates is tracked using a fluorescent marker system. This system allows serial examination of cells within a hydrogel scaffold and correlation of hypoxic signaling with changes in cell phenotype.

Suivi de signalisation hypoxique dans les agrégats de cellules encapsulées

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal ...

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Bio-ingénierie

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

Mesure longitudinale de la matrice extracellulaire rigidité de tumeur modèles 3D utilisant Microrhéologie de particules de suivi

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

À long terme intravitale immunofluorescence imagerie de tissus composants matriciels avec épifluorescence et deux microscopie biphotonique

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

En cartographie in situ des propriétés mécaniques des biofilms par Microrhéologie de particules de suivi

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Plate-forme microfluidique avec multiplexée Détection électronique pour le suivi spatial des particules

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

Montage et suivi du développement de la communauté microbienne dans une plateforme de microwell Array

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

Un protocole pour un suivi en temps réel 3D seule particule

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

Une orbitale secouant la Culture des cellules de mammifères dans les vaisseaux en forme de O pour produire des agrégats uniformes

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

Segmentation de la croissance des cellules endothéliales dans des plaques à 6 puits sur un agitateur orbital pour les études mécanobiologiques

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is catheter-directed thrombolytics (CDT). To mitigate caustic side effects and the long treatment time associated with CDT, adjuvant and alternative approaches are under development. One such approach is histotripsy, a focused ultrasound therapy to ablate tissue via bubble cloud nucleation. Pre-clinical studies have demonstrated strong synergy between histotripsy and thrombolytics for clot degradation. This report outlines a benchtop method to assess the efficacy of histotripsy-aided thrombolytic therapy, or lysotripsy.
Clots manufactured from fresh human venous blood were introduced into a flow channel whose dimensions and acousto-mechanical properties mimic an iliofemoral vein. The channel was perfused with plasma and the lytic&#160;recombinant tissue-type plasminogen activator. Bubble clouds were generated in the clot with a focused ultrasound source designed for the treatment of femoral venous clots. Motorized positioners were used to translate the source focus along the clot length. At each insonation location, acoustic emissions from the bubble cloud were passively recorded, and beamformed to generate passive cavitation images. Metrics to gauge treatment efficacy included clot mass loss (overall treatment efficacy), and the concentrations of D-dimer (fibrinolysis) and hemoglobin (hemolysis) in the perfusate. There are limitations to this in vitro design, including lack of means to assess in vivo side effects or dynamic changes in flow rate as the clot lyses. Overall, the setup provides an effective method to assess the efficacy of histotripsy-based strategies to treat DVT.

Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.