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

1. Probenvorbereitung <ol><li> Aufrechterhaltung CHOK1-Zellen in Gewebekulturflaschen mit DMEM mit 10% fötalem Rinderserum und 100 IU / ml Penicillin 50 ug / ml Streptomycin ergänzt. Die Zellen in einem 5% CO 2 befeuchteten Inkubator bei 37 ° C aufweist. </li><li> Ernte und dann die Platte CHOK1 Zellen auf einem 14 mm Durchmesser Mikro gut mit einer Oberfläche Dicke von 0,16 mm. Samenzellen für eine optimale Dichte für die Bildgebung, etwa 60-70% Konfluenz. </li><li> Zellen bei 37 ° C über Nacht inkubieren, 5% CO 2. In HBSS (Hank Kochsalzlösung), waschen die Zellen dreimal, und Inkubation Zellen in einer Lösung, die 50 nM Lysotracker DND26 grün und 150 nm von Tubulin-Tracker grün. Zellen Inkubation für 1 h bei 37 ° C aufweist. Optionaler Schritt: Vor der Inkubation, führen Sie eine zusätzliche Fleck mit einem mitochondrialen Matrix Färbung Farbstoff (25 nM). </li><li> Waschen Sie die Zellen dreimal in HBSS um ungebundenen Farbstoff zu entfernen. Fügen Sie frisches DMEM Wachstumsmedium vor der IMAGing die Zellen. </li></ol> 2. Mikroskop Konfigurationen Das Mikroskop für die in der Video-Protokoll beschrieben Partikelverfolgung auf dem Rahmen eines im Handel erhältlichen Umkehrmikroskop (4) zusammengebaut. Allerdings kommerziellen Module für 3D-Orbitalpartikelverfolgung sind jetzt verfügbar. <ol><li> Verwenden Sie ein Coherent Chameleon Ultra II-Ti: Sa Femtosekunden-Laseranregungslichtquelle, mit einer abstimmbaren Ausgangswellenlängenbereich zwischen 690 nm-1040 nm auf. </li><li> Sicherzustellen, dass der Laserstrahl in der hinteren Öffnung des Mikroskops unter Verwendung von IR-beschichtete Spiegel ausgerichtet ist. Der Laserstrahl wird mittels eines rotierenden Halbwellenplatte, gefolgt von einem linearen Polarisator Calcit gedämpft. Dämpfen den Strahl, so dass die mittlere Leistung an der Probe zwischen 0,5 und 2 mW. HINWEIS: Der Laserstrahl wird durch ein Paar von Galvanometer-Stellspiegeln, die Motorsteuerung der Position und der Trajektorie des fokussierten Strahls in der Probenebene reflektierte ermöglichen. In einetypische Konfiguration der kollimierte Laserstrahl-Austritts der Galvanometerspiegel expandiert (10x), die durch einen Strahlexpander, bevor sie in die Rückseite des Mikroskopobjektiv nach Reflexion am Kurzpass dichroitischer Spiegel. </li><li> Sammeln Sie das Fluoreszenzlicht aus der Probe, indem eine hohe numerische Apertur Wasser Objektiv (60x, NA 1.2) in den Strahlengang. Wähle ein Fluoreszenzfilterwürfel gemäß den gewünschten Emissionswellenlängen in einer oder beiden Kanalkonfiguration. Beschäftigen Emissionsbandpassfilter weiter wählen den Spektralbereich der emittierten Fluoreszenz. </li><li> Legen Sie die Probe auf einen motorisierten Tisch, und stellen Sie die Feinbewegung des Mikroskopobjektivs mit Hilfe einer objektiven Piezo-Controller. </li><li> Lenken das Licht von der Filterwürfel in Photovervielfacherröhren, wo das Signal diskriminiert und in ein digitales I / O-Datenerfassungskarte gesendet. HINWEIS: Computer erzeugten Wellenformen sind das analoge Ausgangssignal des I / O-Karte und provided den Scanner Steuerelektronik. Die Photonenzählung Eingabe von den Photomultipliern und das Ausgangssignal an den Scanner gemessen und über die I / O-Karte durch das Labor für Fluoreszenzdynamik SimFCS Software gesteuert. </li></ol> 3. Imaging <ol><li> Positionieren Sie die Zellen, die auf der Bühne und konzentrieren sich mit Durchlichtbeleuchtung. </li><li> Wechseln Sie zu Raster-Scan-Bildgebung, um die Zellen, die den Farbstoff aufgenommen haben zu identifizieren und die zugrunde liegenden Mikrotubuli zu visualisieren. Identifizieren Sie die erste Bereich, in dem Vesikel Bewegung vorhanden ist. </li><li> Sobald eine isolierte Vesikel identifiziert wird, legen Sie die folgenden Parameter für die Orbitalverfolgung: <ol><li> Wählen des Radius der Umlaufbahn, um die Größe der Kreisabtastung entsprechend der Größe der Teilchen verfolgt definieren. Für einen Punkt Emitter, stellen Sie den Radius der Umlaufbahn gleich der Taille des Point Spread Function (PSF) der Anregungsstrahl, um die Empfindlichkeit und Reaktion zu maximieren. </li><li> Stellen Sie die axialeAbstand zu dem Abstand zwischen den beiden Bahnen, die ausgeführt werden, um die Partikelposition in der axialen Richtung zu lokalisieren definieren. Stellen Sie den axialen Abstand zu 1,5-3 mal der PSF Taille. </li><li> Definieren Sie die Verweildauer je nach der Helligkeit des Teilchens, die Zeit auf jedem Punkt der Umlaufbahn, die auch die Foto-Bleichrate bestimmen verbrachte eingestellt. Verwenden Sie eine Verweildauer zwischen 10-100 us. </li><li> Die Anzahl der Punkte in jeder Umlaufbahn auf 64 oder 128 in den Orbit Perioden in der Größenordnung von 4-32 ms ergeben und bieten eine hohe zeitliche Auflösung für die Bestimmung der Partikelposition. </li></ol></li><li> Ändern des Gleichstromoffsetsignals an den Spiegel, um den Strahl auf den Teilchen durch die grafische Benutzerschnittstelle über eine Cursor-Auswahl in dem gerasterten Bild Zentrum geschickt. </li><li> Beginnen Tracking durch Umschalten des Mikroskop-Modus von Rasterbildern wird Orbital Scannen. </li><li> Aktivieren Sie die Feedback-und Datenerfassung. </li></ol> 4. Trajectcher Analyse <ol><li> Verwenden Sie die Software, um die an jedem Punkt entlang der Bahn und zu jedem Zeitpunkt in der Form einer &quot;Intensität Teppich &#39;gesammelt Fluoreszenz anzuzeigen. Die entlang der Teppich gesammelt Intensität Informationen über die Wechselwirkung des Teilchens mit anderen hellen Objekten. Die Software verwenden, um sowohl das Bahninformationen (über die Zeit der x, y-Scanner und der z -piezo dh die Verschiebung DC) als auch die Fluoreszenzintensität in Form von Zeitreihen von der Fotovervielfacherröhre über die Zeit gesammelt wurden. </li><li> Nutzen Sie die Zeit und die Reihendarstellung &#39;Intensität Teppich&#39; Informationen, die nur eine Region von Interesse in der Flugbahn zu wählen. </li><li> Verwenden Sie die Software, um eine 2D-Projektion des ausgewählten Teil der Partikelflugbahn an. Wählen Sie die entsprechenden Kontrollen Farbcode die Flugbahn nach der Partikelfluoreszenzintensität. </li><li> Wählen Sie die Option, um die p angezeigtArtikel Bahn in 3D, und Farbcode es nach der Fluoreszenzintensität. Drehen Sie die Flugbahn in 3D über die Steuerelemente, um die Funktionen des Lysosom Bewegung entlang der Mikrotubuli zu visualisieren. </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.

Trotz der enormen Fortschritte der Fluoreszenzmikroskopie-Techniken und Instrumente in den letzten Jahren erreichen Bahnen der fluoreszierenden Teilchen in drei Dimensionen mit einer hohen zeitlichen Auflösung ist eine Herausforderung im Bereich geblieben. Wenn hohe zeitliche Auflösung wurde Tracking Teilchen in zwei Dimensionen erreicht, die Erweiterung zu der axialen Richtung führt typischerweise zu einer drastischen Reduktion der Frequenzgang des Systems 10. In diesem Video-...

Zahlreiche Ansätze sind bisher entwickelt worden, um fluoreszierende Partikel in drei Dimensionen unter Verwendung eines Mikroskops zu verfolgen. Die meisten Ansätze beruhen auf der Verwendung von schnellen Kameras ideal geeignet, um in zwei Dimensionen, die typischerweise mit individuellen Änderungen der Emissionsoptik des Mikroskops kombiniert Verfolgung in axialer Richtung zu erreichen verfolgen. Laser-Scanning-Mikroskop (entweder konfokale oder zwei Photonen) üblicherweise eine Leuchtstoffpartikel durch Ausführen einer zeitlichen Abfolge von Z-Stapeln zu verfolgen, wobei dieses Verfahren typischerweise zeitaufwendig und liefert angemessene zeitliche Auflösung (10 Hz), wenn das Teilchen verfolgt wird in der Mitte eines kleinen Rasterabbildungsbereich durch eine aktive Rückkopplungsmechanismus 2 gehalten. Die Idee der Verriegelungs, um die Partikel der Boden der Orbitalverfolgungsverfahren. Anstelle von einer Rasterabtastung einer Kreisbahn um den fluoreszierenden Teilchen durchgeführt. Die Intensität der Fluoreszenz entlangdie Umlaufbahn genau lokalisiert die Partikelposition 4. Die lokalisierte Position des Partikels kann dann verwendet werden, um die Mikroskopscanner und Wieder Zentrum der Umlaufbahn von der Partikelposition zu betätigen. Die Galvanometer-Scannern des Mikroskops durch eine analoge Spannung angetrieben. Die Wechselstromkomponente dieser Spannung ermöglicht die Durchführung einer Umlaufbahn mit dem fokussierten Laserstrahl, dh einer Sinus und einer zur X und Y Scanspiegel angewendet wird es Durchführen einer kreisförmigen Umlaufbahn Kosinuswelle. Der Gleichstrom-Offset des Signals erleichtert das Ändern der Position der Bahn entfernt. Sobald eine Umlaufdauer bestimmt wird und die Wellenform des Wechselspannungssignals konfiguriert ist, muss das Rückführsystem, um nur die DC-Komponente des Signals zu aktualisieren. Eine Software in der Lage, die von den Detektoren gesammelten Fluoreszenzsignal ausgelesen wird benötigt, um die Scanner und den Detektoren zu steuern, um die Position des Teilchens zu berechnen und zu aktualisieren, die DC ausgesetzt. Für eine erfolgreiche Rückmeldung Abbilden einer sorgfältigen Auswahl der Bahndaten (Größe und Timing) erforderlich ist, und diese Parameter auf der Basis der Eigenschaften der Leuchtstoffpartikel, die es notwendig, zu verfolgen ist, eingestellt werden. Die physikalischen und mathematischen Grundlagen der Technik wurden in der Vergangenheit 4,5 für 2D-und 3D-Anwendungen beschrieben. In diesem Protokoll beschreiben wir kurz die wesentlichen Hardware-Komponenten der Einrichtung, und genauer die Wahl der Parameter und die Probenvorbereitung für einen typischen Versuch erlaubt uns folgende Verschiebung eines Lysosom mit hoher zeitlicher Auflösung in einer lebenden Zelle erforderlich.

<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

Das Ziel dieses Video-Protokoll ist zu diskutieren, wie mit einer modifizierten Zwei-Photonen-Mikroskop 1 durchzuführen und zu analysieren, eine dreidimensionale Fluoreszenzorbitalpartikelverfolgung Experiment. Wie zu herkömmlichen Ansätzen (Rasterabtastung oder Weitfeld basierend auf einem Stapel von Frames) gegenüberliegt, kann die 3D-Tracking-Orbital zu lokalisieren und zu verfolgen mit hoher räumlicher (10 nm Genauigkeit) und zeitliche Auflösung (50 Hz Frequenzgang) die 3D-Verschiebung ein bewegtes fluoreszierende Partikel auf Längenskalen von Hunderten von Mikrometern 2. Das Verfahren basiert auf einem Algorithmus, der die Rückkopplungs Hardware eines Zwei-Photonen-Laser-Scanning-Mikroskop, um eine Kreisbahn um das Objekt auszuführen steuert basierend verfolgt werden: Der Rückkopplungsmechanismus wird das fluoreszierende Objekt in der Mitte durch die Steuerung der Verschiebung zu halten der Abtaststrahl 3-5. Um die Vorteile dieser Technik zu demonstrieren, folgten wir einem sich schnell bewegenden Organelle, das Lysosom, in aliving Zelle 6,7. Zellen wurden nach Standardprotokollen überzogen, und unter Verwendung eines im Handel Lysosom Farbstoff. Wir besprechen kurz die Hardware-Konfiguration und im Detail die Steuer-Software, um eine 3D-Orbital Tracking-Experiment in lebenden Zellen durchzuführen. Diskutieren wir im Detail die um die Rastermikroskop zu steuern und zu ermöglichen, die Bewegung des Strahls in einer geschlossenen Bahn um die Teilchen erforderlichen Parameter. Wir schließen, durch den Nachweis, wie diese Methode effektiv verwendet, um die schnelle Bewegung eines markierten Lysosom an Mikrotubuli in 3D in einer lebenden Zelle zu verfolgen. Lysosomen mit Geschwindigkeiten im Bereich von 0,4-0,5 um / s bewegen, in der Regel Anzeige einer gerichteten Bewegung entlang der Mikrotubuli-Netzwerk 8.

Nach diesem Protokoll schnelle 3D-Einzelpartikelverfolgung kann in lebenden Zellen mit einer modifizierten Zwei-Photonen-Mikroskop, um die Verschiebung des Fluoreszenz-markierten Lysosomen verfolgen durchgeführt werden. Die durchgeführte Experiment besteht aus einer isolierten Verfolgung Lysosom bewegenden im Inneren der Zelle nach dem Endosom Reifungsprozess 9. Die Lysosomen wurden mit einem fluoreszierenden grünen Farbstoff angefärbt und bei 930 nm zu nutzen 2-Photonen-Anregung angeregt wird. Unsere Dat...

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.

3D-Tracking-Orbital in einer modifizierten Zwei-Photonen-Mikroskop: einen Antrag bei der Verfolgung von intrazellulären Vesikeln

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 Aufrufe.  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. 

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

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.

Längs Messung der extrazellulären Matrix Steifigkeit in 3D Tumormodelle mit Particle-Tracking Mikrorheologie

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

Forschung

Biotechnik

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.

In-situ-Mapping der mechanischen Eigenschaften von Biofilmen durch Partikel-Tracking Mikrorheologie

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.

Mikrofluidik-Plattform mit Multiplexed elektronische Sensoren für Raumverfolgung von Partikeln

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.

Zusammenbau und Verfolgung der mikrobiellen Gemeinschaftsentwicklung innerhalb einer Microwell Array Plattform

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.

Ein Protokoll für Echtzeit-3D Einzelkorn-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 ...

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.

Orbital schütteln Kultur von Säugerzellen in O-förmigen Schiffe, einheitliche Aggregate zu produzieren

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.

Segmentierung des Wachstums von Endothelzellen in 6-Well-Platten auf einem Orbitalschüttler für mechanobiologische Studien

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.

Ein In-vitro-System zur Messung der thrombolytischen Wirksamkeit von Histotripsie und einem lytischen Medikament

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is ...

Improving Reproducibility to Meet Minimal Information for Studies of Extracellular Vesicles 2018 Guidelines in Nanoparticle Tracking Analysis

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many consider that NTA instruments and their software packages can be easily utilized following minimal training and that size calibration is feasible in-house. As both NTA acquisition and software analysis constitute EV characterization, they are addressed in Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018). In addition, they have been monitored by Transparent Reporting and Centralizing Knowledge in Extracellular Vesicle Research (EV-TRACK) to improve the robustness of EV experiments (e.g., minimize experimental variation due to uncontrolled factors).
Despite efforts to encourage the reporting of methods and controls, many published research papers fail to report critical settings needed to reproduce the original NTA observations. Few papers report the NTA characterization of negative controls or diluents, evidently assuming that commercially available products, such as phosphate-buffered saline or ultrapure distilled water, are particulate-free. Similarly, positive controls or size standards are seldom reported by researchers to verify particle sizing. The Stokes-Einstein equation incorporates sample viscosity and temperature variables to determine particle displacement. Reporting the stable laser chamber temperature during the entire sample video collection is, therefore, an essential control measure for accurate replication. The filtration of samples or diluents is also not routinely reported, and if so, the specifics of the filter (manufacturer, membrane material, pore size) and storage conditions are seldom included. The International Society for Extracellular Vesicle (ISEV)&#39;s minimal standards of acceptable experimental detail should include a well-documented NTA protocol for the characterization of EVs. The following experiment provides evidence that an NTA analysis protocol needs to be established by the individual researcher and included in the methods of publications that use NTA characterization as one of the options to fulfill MISEV2018 requirements for single vesicle characterization.

Nanoparticle tracking analysis (NTA) is a widely used method to characterize extracellular vesicles. This paper highlights NTA experimental parameters and controls plus a uniform method of analysis and characterization of samples and diluents necessary to supplement the guidelines proposed by MISEV2018 and EV-TRACK for reproducibility between laboratories.

Verbesserung der Reproduzierbarkeit, um minimale Informationen für Studien zu extrazellulären Vesikeln zu erfüllen Richtlinien 2018 in der Nanopartikel-Tracking-Analyse

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many ...

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

Im lebenden Organismus Bildgebung von biologischem Gewebe mit kombinierter Zwei-Photonen-Fluoreszenz und stimulierter Raman-Streumikroskopie

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...