Die Arbeit der Z-und Y-Yang Wang wurde möglich durch Gewährung K02 AR051181 aus NIAMS / NIH, Zuschüsse aus Muscular Dystrophy Association und Harvard Stem Cell Institute for Y Wang. Die Arbeit von Q Zeng und X Xu sind durch ein Programm Zuschuss für das Optical Imaging Lab aus dem Brigham and Women Hospital unterstützt. Die Autoren bedanken sich bei Wen Liu aus der Abteilung für Anästhesie, BWH, danke für die technische Hilfe in der Zelle arbeiten.

Zellpräparation <ol><li> Satellite Zellisolation <ol style="list-style-type:lower-alpha;"><li> Unter Narkose werden die Beinmuskeln der GFP transgenen Mäusen (C57BL / Ka-βactin-EGFP) entfernt. Nach dem Schneiden in kleine Stücke, sind Satelliten-Zellen enzymatisch gespalten durch Inkubation des zerkleinerten Gewebes mit 2% Dispase Kollagenase-Lösung für 1 Stunde bei 37 ° C. </li><li> Neutralisieren das Enzym die Verdauung mit einem Wachstum Medien mit Ham F-10 und 20% FBS, distanzieren den Muskel durch wiederholtes Verreiben mit einer Pasteurpipette. Die Zellen werden durch eine Zelle Sieb (ø70-100 um) filtriert und dann auf einem nicht-beschichteten Teller für 1 Stunde bei 37 ° C beschichtet </li><li> Entfernen Sie vorsichtig den Überstand und die Platte sie auf einer Kollagen-beschichtete Schale. Wiederholen Sie diesen Schritt 1 Stunde später an die Myoblasten zu reinigen. (Pre-Platte) </li><li> Ändern Sie den Medien-und Platte die Zellen in der Zeit. Die endgültige Reinigung Myoblasten werden in Medium mit Ham F-10, 20% FBS, 100 U / ml Penicillin und 100 ug / ml Streptomycin (Wachstumsmedium) bei 37 ° C mit 5% CO2, 95% Luft kultiviert. </li></ol> </li><li> Kultur und erweitern Sie den primären Myoblasten in Wachstumsmedium mit Zugabe basischer FGF von 10 ng / ml. </li><li> Für alle die in-vivo-Experimente, spritzen wir 5x10 5 Zellen pro TA Muskel. </li><li> Bei der Ernte die Myoblasten, lösen wir die Zellen mit 0,25% Trypsin / EDTA und waschen Sie sie mit PBS zweimal. Die Zellzahlen sind mit einer Zählkammer gezählt und die Zellen in HBSS bei einer Konzentration von 5x10 4 Zellen / ul resuspendiert. Die konzentrierte Zellen in HBSS auf Eis gestellt und injiziert innerhalb von 1 Stunde nach der Entnahme. </li></ol> In Vivo Imaging <ol><li> Männlich mdx-Mäusen im Alter von 4-6 Wochen aus Jackson Labor gekauft und bei einem Tier Wohnanlage. </li><li> Nach ca. 1 Woche Unterkunft für die Umwelt, werden die Mäuse bereit, mit den kultivierten Zellen implantiert werden. </li><li> Vor-Transplantation ist die Maus durch eine intraperitoneale (ip) Injektion der Mischung von Ketamin (100 mg / kg) und Xylazin (10 mg / kg) narkotisiert. Dann werden die Haare um den TA Muskel-Bereich wird durch die Anwendung Nair, um das Bein und wartet 30 Sekunden, um wischen Sie sie mit destilliertem Wasser reinigen entfernt. </li><li> Während sich die Maus noch in Narkose, sind 5x10 5 Zellen in 10 &amp;mgr; l HBSS langsam in jeder TA Muskel an 3 Punkten mit einem 30-Gauge-Nadel injiziert. </li><li> Nach Schritt 4, kann die Maus regelmäßig durch in vivo Fluoreszenz-Imaging abgebildet werden. Die Fluoreszenz-Imaging-Station Nightowl LB981 (Berthold Technologies, Inc.) mit einer hochempfindlichen CCD-Kamera ausgestattet, wird verwendet, um Bilder aus dem behindern Beine der Mäuse zu erhalten. </li><li> Am Tag des In-vivo-Bildgebung, überprüfen wir zunächst die das Nachwachsen der Haare auf dem TA Muskel. Wenn nötig, werden wir erneut bewerben Nair, um das Haar, wie oben beschrieben zu entfernen, nachdem Anästhesie der Maus mit der Mischung aus Ketamin und Xylazin. </li><li> Während sich die Maus in Narkose legen wir die Maus in Bauchlage auf einem Stück schwarzen, nicht fluoreszierendem Papier (schwarz Artagain Papier aus Strathmore). Um vollständig aussetzen TA Muskel für eine bessere Bildgebung, tape wir die Hinterpfoten, um das Papier. </li><li> Wir platzieren Sie den Mauszeiger in der Imaging-Kammer und die Position der Hinterbeine, um in der Mitte des Sichtfeldes der Kamera abgebildet werden. Wir ersten Schalter aus dem Emissions-Filter zu einem traditionellen Graustufen-Foto-Bild des Beines mit einer Belichtungszeit von 5 Sekunden dauern. Danach ist die Position der Maus und Fokus der Kamera, wenn nötig. Die Imaging-Parameter einschließlich Belichtungszeit (1.250 ms), Probenhöhe (0,9 cm) etc. konstant gehalten werden in der experimentellen Verfahren. </li><li> Wir haben dann in die entsprechende Emission Filter-Schalter für das grün fluoreszierende Protein Wellenlänge. Wir nehmen Fluoreszenzbild mit einer Belichtungszeit von 1.250 ms und einem Binning-Zahl von 1. Nach der Belichtung wird die Maus aus dem Imaging-Kammer auf einen Käfig, um Erholung zu warten entfernt. </li><li> Für die Analyse erstellen wir eine Region of Interest (ROI) über die Fluoreszenz-Signale ihrer Intensität in Bezug auf die Photonen pro Sekunde zu messen. Wir messen auch die Größe der ROI in Bezug auf die Anzahl der Pixel als eine weitere Quantifizierung Wert. Die Imaging-Experimente können jede Woche wiederholt werden oder häufiger über ein paar Monate, falls erforderlich, Längsschnittdaten zu erwerben. </li></ol> Am Ende der Experimente wird die Maus getötet und das TA Muskel ist für die Histologie Analyse geerntet.

<ol>
	<li>Ballou, B., Ernst, L. A., Waggoner, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+imaging+of+tumors+in+vivo.">Fluorescence imaging of tumors in vivo.</a> Curr Med Chem. 12, 795-805 (2005).</li><li>Green, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fluorescent+protein+as+a+marker+for+gene+expression.">fluorescent protein as a marker for gene expression.</a> Science. 263, 802-805 (1994).</li><li>Godavarty, A., Sevick-Muraca, E. M., Eppstein, 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=Three-dimensional+fluorescence+lifetime+tomography.">Three-dimensional fluorescence lifetime tomography.</a> Med Phys. 32, 992-1000 (2005).</li><li>Graves, E. E., Weissleder, R., Ntziachristos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+molecular+imaging+of+small+animal+tumor+models.">Fluorescence molecular imaging of small animal tumor models.</a> Curr Mol Med. 4, 419-430 (2004).</li><li>Graves, E. E., Yessayan, D., Turner, G., Weissleder, R., Ntziachristos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+in+vivo+fluorochrome+concentrations+measured+using+fluorescence+molecular+tomography.">Validation of in vivo fluorochrome concentrations measured using fluorescence molecular tomography.</a> J Biomed Opt. 10, 44019-44 (2005).</li><li>Gurfinkel, M., Ke, S., Wen, X., Li, C., Sevick-Muraca, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+fluorescence+optical+imaging+and+tomography.+Dis+Markers.">Near-infrared fluorescence optical imaging and tomography. Dis Markers.</a> , 107-121 .</li><li>Lee, J., Sevick-Muraca, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+fluorescence+enhanced+optical+tomography+using+referenced+frequency-domain+photon+migration+measurements+at+emission+and+excitation+wavelengths.">Three-dimensional fluorescence enhanced optical tomography using referenced frequency-domain photon migration measurements at emission and excitation wavelengths.</a> J Opt Soc Am A Opt Image Sci Vis. 19, 759-771 (2002).</li><li>Xu, X., Yang, Z., Liu, Q., Wang, Y. <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+fluorescence+imaging+of+muscle+regeneration+by+transplanted+eGFP-labeled+myoblasts.">In vivo fluorescence imaging of muscle regeneration by transplanted eGFP-labeled myoblasts.</a> , (1985).</li><li>Hoffman, R. M. <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+cell+biology+of+cancer+cells+visualized+with+fluorescent+proteins.">In vivo cell biology of cancer cells visualized with fluorescent proteins.</a> Curr Top Dev Biol. 70, 121-144 (2005).</li><li>Coulton, G. R., Morgan, J. E., Partridge, T. A., Sloper, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mdx+mouse+skeletal+muscle+myopathy:+I.+A+histological,+morphometric+and+biochemical+investigation.">The mdx mouse skeletal muscle myopathy: I. A histological, morphometric and biochemical investigation.</a> Neuropathol Appl Neurobiol. 14, 53-70 (1988).</li><li>Morgan, J. E., Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+satellite+cells.">Muscle satellite cells.</a> Int J Biochem Cell Biol. 35, 1151-116 (2003).</li><li>Mendell, J. R., Kissel, J. T., Amato, A. A., King, W., Signore, L., Prior, T. W., Sahenk, Z., Benson, S., McAndrew, P. E., Rice, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myoblast+transfer+in+the+treatment+of+Duchenne's+muscular+dystrophy.">Myoblast transfer in the treatment of Duchenne's muscular dystrophy.</a> N Engl J Med. 333, 832-838 (1995).</li><li>Bogdanovich, S., Perkins, K. J., Krag, T. O., Khurana, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutics+for+Duchenne+muscular+dystrophy:+current+approaches+and+future+directions.">Therapeutics for Duchenne muscular dystrophy: current approaches and future directions.</a> J Mol Med. 82, 102-115 (2004).</li><li>Morgan, J. E., Coulton, G. R., Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mdx+muscle+grafts+retain+the+mdx+phenotype+in+normal+hosts.">Mdx muscle grafts retain the mdx phenotype in normal hosts.</a> Muscle Nerve. 12, 401-409 (1989).</li><li>Sherwood, R. I., Christensen, J. L., Weissman, I. L., Wagers, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+skeletal+muscle+contributions+from+circulating+cells,+bone+marrow+cells,+and+hematopoietic+stem+cells.">Determinants of skeletal muscle contributions from circulating cells, bone marrow cells, and hematopoietic stem cells.</a> Stem Cells. 22, 1292-1304 (2004).</li><li>Cerletti, M., Jurga, S., Witczak, C. A., Hirshman, M. F., Shadrach, J. L., Goodyear, L. J., Wagers, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient,+functional+engraftment+of+skeletal+muscle+stem+cells+in+dystrophic+muscles.">Highly efficient, functional engraftment of skeletal muscle stem cells in dystrophic muscles.</a> Cell. 134, 37-47 (2008).</li></ol>

Wir haben eine zuverlässige und stabile Imaging-Plattform für die Verfolgung der fluoreszenzmarkierten transplantierten Zellen in Host Skelettmuskulatur in dieser Studie. GFP-markierten Myoblasten aus GFP transgenen Maus steht für einen Typ von adulten Stammzellen, die Kandidaten für die Zelltherapie in der Zukunft sein wird. Eine ständige Manipulation einschließlich Zellzahl, Zell-Injektion, klaren Hintergrund, Bildposition und Maschinen-Parameter ist für die Gewinnung hochwertiger Bild-und vor allem für die quantitative Analyse wichtig. Fluoreszenz-Bildgebung hat viele Anwendungen in der biomedizinischen Forschung, wie sie nicht-invasiv, einfach zu bedienen ist, und hat einen hohen Durchsatz. Neben Fluoreszenz-Bildgebung kann eine quantitative Messung der Photonen, obwohl aufgrund der Streuung, Dämpfung und Absorption basiert bereitzustellen, kann Licht nicht durchdringen einen großen Abstand im Gewebe, die einen Mangel der Technik ist. Es wurden Anstrengungen im Gange, um Rot-oder Nah-Infrarot-Reporter nutzen, um die Eindringtiefe des Lichts zu erhöhen. Ein weiterer Vorteil der Fluoreszenz-Bildgebung ist, dass es übersetzbar Kliniken ist, wenn die spezifischen fluoreszierenden Reporter für den menschlichen Gebrauch zugelassen ist. Im Vergleich mit MRT, CT und PET, hat Fluoreszenz-Bildgebung eine hohe Flexibilität, da sie nicht teure Hardware-und Imaging-Agenten. Ein Beispiel hierfür ist Fluoreszenz-basierten Endoskop und Mikro-Endoskop, das in Kliniken verwendet wurden. Durch die Kombination von Fluoreszenz-Imaging mit anderen Modalitäten, erwarten wir die Fähigkeit, sowohl anatomische und funktionelle Informationen über die zugrunde liegenden biologischen Prozesse erwerben zu erreichen.

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<td>NightOwl LB981</td>
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tracking dynamics of muscle engraftment in small animals by in vivo fluorescent imaging

Muskeldystrophien sind eine Gruppe von degenerativen Muskel-Erkrankungen, die durch fortschreitenden Verlust der kontraktilen Zellen gekennzeichnet. Derzeit gibt es keine kurative Behandlung nicht möglich. Jüngste Fortschritte in der Stammzellbiologie haben neue Hoffnungen für die Entwicklung wirksamer Therapien Zelle erzeugt werden, um diese Krankheiten zu behandeln. Transplantation von verschiedenen Arten von Stammzellen mit fluoreszierenden Proteine ​​in Muskeln der dystrophischen Tiermodellen beschriftet wurde weitgehend in das Feld verwendet. Eine nicht-invasive Technik mit der Möglichkeit, die transplantierten Zellen Schicksal track längs können weiter unsere Fähigkeit, Muskel-Transplantation von transplantierten Zellen genauer und effizienter zu bewerten. In der vorliegenden Studie zeigen wir, dass in vivo Fluoreszenz-Imaging ist eine sensible und zuverlässige Methode für die Verfolgung von transplantierten GFP (Green Fluorescent Protein)-markierten Zellen in Maus Skelettmuskeln. Trotz der Besorgnis über die Hintergründe durch die Verwendung eines externen Licht notwendig für die Anregung des fluoreszierenden Proteins, fanden wir, dass, indem Sie entweder Nacktmaus oder Beseitigung Haar mit Haarentfernung Reagenzien viel von diesem Problem beseitigt ist. Mit einer CCD-Kamera, kann das Fluoreszenzsignal in den Tibialis anterior (TA) Muskel nach der Injektion von 5 x 10 5 Zellen entweder von GFP transgenen Mäusen oder eGFP transduziert Myoblasten Kultur erkannt werden. Für weitere oberflächliche Muskeln wie dem M. extensor digitorum longus (EDL), Injektion von weniger Zellen produziert ein nachweisbares Signal. Signalintensität gemessen und quantifiziert werden, da die Anzahl der emittierten Photonen pro Sekunde in einer region of interest (ROI). Da die aufgenommenen Bilder klare Grenzen zur Abgrenzung der transplantierten Bereich zeigen, kann die Größe der ROI sowie gemessen werden. Wenn die Beine konsequent jedes Mal positioniert sind, spiegeln die Veränderungen in der Gesamtzahl der Photonen pro Sekunde pro Muskelgruppe und die Größe der ROI die Änderungen in der Zahl der transplantierten Zellen und die Größe des eingepflanzten Bereich. Die Veränderungen in den gleichen Muskel im Laufe der Zeit sind quantifizierbar. In vivo Fluoreszenz-Imaging-Technik wurde in erster Linie auf das Wachstum von Zellen tumorogene Spur verwendet, unsere Studie zeigt, dass es ein mächtiges Werkzeug, das uns ermöglicht, das Schicksal der transplantierten Stammzellen Weg ist.

Watch this Scientific Journal Video about Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging at JoVE.com

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Wir beschreiben einen<em&gt; In vivo</em&gt; Fluoreszenz-Imaging-Protokoll, um die Regeneration der Muskulatur von GFP-markierten Myoblasten Monitor nach der Transplantation in der Skelettmuskulatur von gesunden und dystrophischen Mäusen. Dieses Protokoll kann angepasst an die Regeneration der Muskulatur durch die Transplantation von anderen Arten von Zellen und in anderen Muskelerkrankungen sowie zu studieren.

Tracking-Dynamics of Muscle Engraftment bei Kleintieren durch In Vivo Fluorescent Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

tracking-dynamics-muscle-engraftment-small-animals-vivo-fluorescent

Research

JoVE Journal

Biology

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

9.7K Aufrufe.  Brigham and Woman's Hospital. Wir beschreiben einen In vivo Fluoreszenz-Imaging-Protokoll, um die Regeneration der Muskulatur von GFP-markierten Myoblasten Monitor nach der Transplantation in der Skelettmuskulatur von gesunden und dystrophischen Mäusen. Dieses Protokoll kann angepasst an die Regeneration der Muskulatur durch die Transplantation von anderen Arten von Zellen und in anderen Muskelerkrankungen sowie zu studieren.

Serie: Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

In-vivo-Mikro-Zirkulation Measurement im Skelettmuskel von Intra-vital-Mikroskopie

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Forschung

Biologie

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

Labeling hESCs und hMSCs mit Eisenoxid-Nanopartikel für Non-Invasive in vivo-Tracking mit MR-Bildgebung

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

Live Cell Imaging von F-Aktin Dynamics über Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

Ex Vivo Beurteilung der Kontraktilität, Müdigkeit und Alternans in isolierten Skelettmuskulatur

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

In vivo Klonale Verfolgung von hämatopoetischen Stamm-und Vorläuferzellen durch Fünf Fluoreszierende Proteine ​​mit Hilfe der konfokalen und Multiphotonen Mikroskopie Markierte

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In-vivo-4-Dimensional-Tracking von hämatopoetischen Stamm-und Vorläuferzellen im erwachsenen Maus Schädeldachknochenmark

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Hochauflösende Zeitraffer-Imaging und automatische Analyse von Dynamik der Mikrotubuli in lebenden menschlichen Endothelzellen aus der Nabelvene

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult mice for in vivo longitudinal real-time monitoring of thymocytes&#180; dynamics within a vascularized thymus segment. Following the transplantation, laser scanning microscopy (LSM) through the cornea allows in vivo noninvasive repeated imaging at cellular resolution level. Importantly, the approach adds to previous intravital T-cell maturation imaging models the possibility for continuous progenitor cell recruitment and mature T-cell egress recordings in the same animal. Additional advantages of the system are the transparency of the grafted area, permitting macroscopic rapid monitoring of the implanted tissue, and the accessibility to the implant allowing for localized in addition to systemic treatments. The main limitation being the volume of the tissue that fits in the reduced space of the eye chamber which demands for lobe trimming. Organ integrity is maximized by dissecting thymus lobes in patterns previously shown to be functional for mature T-cell production. The technique is potentially suited to interrogate a milieu of medically relevant questions related to thymus function that include autoimmunity, immunodeficiency and central tolerance; processes which remain mechanistically poorly defined. The fine dissection of mechanisms guiding thymocyte migration, differentiation and selection should lead to novel therapeutic strategies targeting developing T cells.

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The goal of the protocol is to show longitudinal intravital real-time tracking of thymocytes by laser scanning microscopy in thymic implants in the anterior chamber of the mouse eye. The transparency of the cornea and vascularization of the graft allows for continuously recording progenitor cell recruitment and mature T-cell egress.

Echtzeit- In Vivo Verfolgung von Thymozyten in die Vorderkammer des Auges durch Laser-Scanning-Mikroskopie

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Untersuchung der Mitotic and Meiotic Fission Yeast Nuclear Dynamics durch Fluoreszenz-Livezellmikroskopie

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis. N&#38;B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&#38;B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&#38;B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&#38;B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis.

Oligomerisierungsdynamik von Zelloberflächenrezeptoren in lebenden Zellen durch totale interne Reflexionsfluoreszenzmikroskopie kombiniert mit Zahlen- und Helligkeitsanalyse

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...