Wir danken Dr. Till Böcking für das ursprüngliche Einzelmolekül-Vorbereitungsprotokoll. 
Diese Arbeit wurde durch den NSF-Zuschuss 1121972(SS) unterstützt.

<ol>
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J. 92, 1777-1783 (2007).</li><li>Ortega-Esteban, A., 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=Minimizing+tip&#8211;sample+forces+in+jumping+mode+atomic+force+microscopy+in+liquid.">Minimizing tip&#8211;sample forces in jumping mode atomic force microscopy in liquid.</a> Ultramicroscopy. 114, 56-61 (2012).</li><li>Ortega-Esteban, A., 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=Monitoring+dynamics+of+human+adenovirus+disassembly+induced+by+mechanical+fatigue.">Monitoring dynamics of human adenovirus disassembly induced by mechanical fatigue.</a> Sci. Rep. 3, (2013).</li><li>Hernando-P&#233;rez, M., 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=Physical+Virology:+Direct+Measurement+of+Phage.">Physical Virology: Direct Measurement of Phage.</a> phi29 Stiffness Provides Evidence of Internal Pressure (Small 15/2012). Small. 8, 2365-2365 (2012).</li><li>Carrasco, C., 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=DNA-mediated+anisotropic+mechanical+reinforcement+of+a+virus.">DNA-mediated anisotropic mechanical reinforcement of a virus.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 13706-13711 (2006).</li><li>Sofia, S. J., Premnath, V. V., Merrill, E. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+binding+experiments+on+long+time+scales.">Single-molecule binding experiments on long time scales.</a> Rev. Sci. Instrum. 81, (2010).</li><li>van Oijen, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Molecule+Kinetics+of+&#955;+Exonuclease+Reveal+Base+Dependence+and+Dynamic+Disorder.">Single-Molecule Kinetics of &#955; Exonuclease Reveal Base Dependence and Dynamic Disorder.</a> Science. 301, 1235-1238 (2003).</li><li>B&#246;cking, T., Aguet, F., Harrison, S. C., Kirchhausen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+analysis+of+a+molecular+disassemblase+reveals+the+mechanism+of+Hsc70-driven+clathrin+uncoating.">Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating.</a> Nat. Struct. Mol. Biol. 18, 295-301 (2011).</li><li>Cocucci, E., Aguet, F., Boulant, S., Kirchhausen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+First+Five+Seconds+in+the+Life+of+a+Clathrin-Coated+Pit.">The First Five Seconds in the Life of a Clathrin-Coated Pit.</a> Cell. 150, 495-507 (2012).</li><li>Rust, M. J., Bates, M., Zhuang, X. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-diffraction-limit+imaging+by+stochastic+optical+reconstruction+microscopy.">Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy.</a> 3, 793-795 (2006).</li><li>Betzig, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+intracellular+fluorescent+proteins+at+nanometer+resolution.">Imaging intracellular fluorescent proteins at nanometer resolution.</a> Science. 313, 1642-1645 (2006).</li><li>Hess, S. T., Girirajan, T. P. K., Mason, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-High+Resolution+Imaging+by+Fluorescence+Photoactivation+Localization+Microscopy.">Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy.</a> Biophys. J. 91, 4258-4272 (2006).</li><li>Juette, M. F., 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=Three-dimensional+sub-100+nm+resolution+fluorescence+microscopy+of+thick+samples.">Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples.</a> Nat. 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Die Autoren haben in dieser Zeitung keine konkurrierenden finanziellen Interessen.

Als alternative Methode zur CryoEM-Tomographie kann eine Einzelvirion-Bildgebung mit AFM und hochauflösende Fluoreszenz-Bildgebung verwendet werden. Jede dieser Methoden hat ihre spezifischen Stärken. Zum Beispiel kann AFM auf WT Virionen ohne Anforderung auf Diepging der Probe oder der internen viralen Proteine durchgeführt werden. Die Lage viraler Strukturen ist von ihren Beiträgen zu den elastischen Eigenschaften der Virion entkoppelt.
Single Virion Super-Resolution Imaging in Kombination mit den neu entwickelten viralen Reverse genetischen Ansätzen wird eine leistungsfähige Technik zur Lokalisierung der niedrigen Kopierzahl viraler Proteine20. Rekombinante Viren mit Ersatz ihrer Proteine durch Proteine, die zu fluoreszierenden Proteinen verschmolzen sind, können mit diesen umgekehrten genetischen Ansätzen hergestellt und gereinigt werden. Obwohl die Superauflösungsabbildung zum Teil aufgrund der genetischen Kennzeichnung Spezifität hat, erfordert sie die Verwendung der mutierten Viren.
AFM und Super-Resolution Imaging sind kostenlose Methoden, die sehr ähnliche Probenpräparate verwenden. Die in diesem Papier beschriebenen Methoden, die von früheren Einzelmolekül-Bildgebungs-Assays übernommen werden, ermöglichen die Verankerung einzelner Virionen mit geringen Auswirkungen auf die Topologie der Virionen.

Ladungsbasierte unspezifische Wechselwirkungen werden routinemäßig zur Adhäsion von Virionen in der Atomkraftmikroskopie1,2verwendet. Diese Techniken funktionieren besonders gut, wenn sie auf nicht umhüllten Virionen mit sehr steifen Kapsids3-6verwendet werden. Obwohl diese Techniken sehr effektiv bei der Immobilisierung der Probe sind, verhindern sie nicht die unspezifische Bindung von Proteinen an die Oberfläche. Die unspezifische Bindung kann ein Problem verursachen, wenn versucht wird, Virionen mit AFM und super-Resolution-Fluoreszenztechniken abzubilden, die Inkubationen der Probe mit verschiedenen Antikörpern erfordern. Hier skizzieren wir eine Beispielvorbereitungsmethode für die spezifische Immobilisierung von Virions.
Poly(Ethylenglykol) (PEG) mit Poly(L-Lysin) (PLL) veredelt und auf eine Glasoberfläche adsorbiert stellt einen signifikanten Block für die elektrostatischen Wechselwirkungen von Proteinen mit Glas7. Einzelmolekül-Assays, die eine Immobilisierung einzelner Moleküle auf Glasoberflächen erfordern, haben diese Eigenschaft genutzt und sie verwendet, um eine spezifische PEG-basierte Einzelmolekül-Immobilisierungstechnik8-10zu erstellen. Dieses Präparat wurde auch verwendet, um Clathrinkäfige auf die Glasoberfläche11 zu immobilisieren sowie eine homogene Fibronectin-Beschichtung für die Kontrollzellhaftung 12zu schaffen.
Wir haben das Probenvorbereitungsverfahren auf DerGrundlage der PLL-g-PEG-Adsorption auf Glasoberflächen aus bildgebenden Methoden für einzelne Moleküle übernommen und auf bildgebende Einzelvirionen des vesikulären Stomatitisvirus (VSV) angewendet. Diese einzelnen Virionen wurden mit Hilfe der Atomkraftmikroskopie (AFM) abgebildet. Ähnliche Experimente wurden auch an funktionalisierten Perlen als Kontrolle durchgeführt. Die Virionen wurden auch durch superhochauflösende Fluoreszenzmikroskopie abgebildet, wenn ein VSV-G-Antikörper mit Alexa 647 beschriftet wurde, um ein Bild der Umhüllung einzelner Virionen zu erstellen. Hochauflösende Fluoreszenz-Bildgebung nutzt die Lokalisierung einzelner Moleküle, um ein Bild13-15zu erstellen. fPALM bi-planare Bildgebung ermöglicht die Lokalisierung einzelner Moleküle mit 20 nm in der Ebene und 50 nm Auflösung entlang der optischen Achse15,16. Diese biplanare Technik wurde verwendet, um in dieser Studie enthaltene fluoreszierende Bilder mit superauflösung abzubilden. Eine alternative Technik, die ähnliche Ergebnisse hat, ist STORM13,17,18.
Sowohl AFM als auch superhochauflösende fluoreszierende Bilder von VSV, die durch die in diesem Papier beschriebenen Verfahren am Glas verankert sind, zeigten eine spezifische Bindung von Virionen an das Glas mit minimalen unspezifischen Wechselwirkungen19. Hier stellen wir die Probenvorbereitungsprotokolle für die AFM- und super-resolution fluoreszierenden Bildgebungsexperimente vor. Kurz gesagt: Saubere Glasabdeckungen werden funktionalisiert, indem eine Mischung aus PLL-g-PEG und PLL-g-PEG-Biotin adsorbiert wird. Es ist typisch für diesen dünnen Film, der entwickelt werden kann, um zwischen 15-25% funktionalisiertes Biotin auf einer beschichteten Oberfläche zu liefern. Die Coverlips werden weiter mit tetramerischem Avidin inkubiert. Ein biotinylierter viraler Antikörper wird dann verwendet, um eine einzigartige Bindungsstelle für die Virionen zu schaffen. Die Bindung des Antikörpers kann auf zwei Arten erfolgen.
Die erste Methode ist für AFM optimiert, bleibt jedoch für die Hochauflösende Fluoreszenz-Bildgebung geeignet. Bei diesem Verfahren wird die avidinbeschichtete Oberfläche vor der viralen Adhäsion mit biotinylierten Antikörpern behandelt. Die immobilisierten Virionen werden mit Alexa 647-beschrifteten Antikörpern behandelt, um die Hülle zu bedecken und eine superhochauflösende Fluoreszenz-Bildgebung der Virionen zu ermöglichen.
Die zweite Methode besteht darin, das Virus in Lösung mit dem biotinylierten Antikörper und dem von Alexa 647 markierten Antikörper anzugreifen, bevor die Viren an der mit Avidin behandelten Oberfläche haften; Diese Methode ist für superauflösende Fluoreszenz-Bildgebungsexperimente optimiert, bei denen die Wiederherstellung der viralen Hülle wichtig ist. Dieses Verfahren hat den Vorteil, dass eine gleichmäßige Antikörperbeschichtung auf der viralen Oberfläche möglich ist. Der biotinylierte Antikörper kann mit Alexa 647 als virale Antikörper in Verhältnissen vermischt werden, die eine ausreichende Adsorption des Virus auf die mit Avidin behandelte Oberfläche ermöglichen und gleichzeitig eine hohe Konzentration an fluoreszierendem Etikett an der Außenseite der viralen Hülle beibehalten. Diese Alexa 647 markierten viralen Antikörper sind nicht biotinyliert und ihr Überschuss kann wegspült werden, um Hintergrundgeräusche zu reduzieren.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Glass coverslips (fPALM)</td>
			<td style="width:71px;">Electron Microscopy Services</td>
			<td style="width:119px;">72225-01</td>
			<td style="width:167px;">25 mm Coverslips</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PLL(20)-g[3.5]-PEG(2)/PEG(3.4)-Biotin (20%)&#160;</td>
			<td style="width:71px;">SuSoS&#160;</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">Stock: 0.5 mg/ml in PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NeutrAvidin biotin-binding protein</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">A2666</td>
			<td style="width:167px;">Stock: 0.25 mg/ml in PBS</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Alexa Fluor 647 goat anti-rabbit IgG (H+L)&#160;</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">A21245</td>
			<td style="width:167px;">1:200 in 0.2 M KPO4, 0.15 M NaCl, 10% Glycol, pH 7.2 buffer</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">&#945;-VSV-G&#160;</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">ab34774</td>
			<td style="width:167px;">1:200 in 0.2 M KPO4, 0.15 M NaCl, 10% Glycol, pH 7.2 buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gluox</td>
			<td style="width:71px;">Sigma</td>
			<td>G2133-250KU&#160;</td>
			<td>Glucose oxidase type seven from Aspergillius&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Catalase</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">C40-100mg&#160;</td>
			<td>Catalase from Bovine liver&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEA</td>
			<td style="width:71px;">Sigma</td>
			<td>30070-10G&#160;</td>
			<td>Cysteamine (MEA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stock Buffer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>50 mM Tris-HCl (pH 8.0) + 10 mM NaCl + 10% glucose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NTE</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>10 mM Tris pH 7.4, 100 mM NaCl, 66 mM EDTA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Slide-A-Lyzer, mini dialysis unit</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td>10,000 MW</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Microscope Cover Glass: 35 CIRCLE #1</td>
			<td style="width:71px;">Fisherbrand</td>
			<td style="width:119px;">35 CIRCLE #1</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

sample preparation for single virion atomic force microscopy and super-resolution fluorescence imaging

Die Immobilisierung von Virionen auf Glasoberflächen ist ein entscheidender Schritt in der Einzelvirionen-Bildgebung. Hier stellen wir eine Technik vor, die aus einzelmolekül-Bildgebungs-Assays übernommen wird, die die Haftung einzelner Virionen auf Glasoberflächen mit Spezifität ermöglicht. Diese Zubereitung basiert auf der Veredelung der Glasoberfläche mit einer Mischung aus PLL-g-PEG und PLL-g-PEG-Biotin, der Zugabe einer Avidinschicht und schließlich der Bildung von Virionankern durch Anhaftung biotinylierter virusspezifischer Antikörper. Wir haben diese Technik in einer Reihe von Experimenten angewendet, einschließlich der Atomkraftmikroskopie (AFM) und der superauflösenden Fluoreszenz-Bildgebung. Diese Probenvorbereitungsmethode führt zu einer Kontrollhaftung der Virionen an der Oberfläche.

Einzelvirionen-Bildgebung mit AFM:
Das oben beschriebene Probenvorbereitungsprotokoll wurde bei der Verankerung von Wildartenvirionen an der Glasoberfläche verwendet. VSV-Virionen sind kugelförmig mit einer Länge von 180 nm und einem Durchmesser von 80 nm. Da es eine Vielzahl von Virions gibt, für die diese Technik angewendet werden kann, wird das Konzept auch hier auf biotinylierten 36 nm Perlen demonstriert. Die resultierenden AFM-Experimente sind in Abbildung 1dargestellt. Es ist wichtig zu beachten, dass die VSV-Virionen einen niedrigeren Young-Modul (100 MPa) im Vergleich zu den nicht umhüllten Viren (GPa) haben. Die besonderen Bilder von Single Virion VSV wurden im Abstichmodus unter Umgebungsbedingungen mit einem steifen Ausleger erhalten. Das Ziel war es, das Virus so weit zu verformen, dass die zusätzliche Proteindichte innerhalb des Virus als Beule innerhalb des AFM-Bildes sichtbar wird. Diese Methode wird verwendet, um die zusätzliche Proteindichte innerhalb der Virushöhle19zu erkennen.
Einzelvirion Super Auflösung Bildgebung:
Alexa 647 mit VSV-G-Antikörpern beschriftet wurden verwendet, um die Hülle einzelner VSV-Virionen für Super-Auflösungsexperimente mit Methode 5A zu beschichten. Um die Tetheringdichte und die geringe unspezifische Bindung zu veranschaulichen, ist in Abbildung 2Aein großer Scan der Probe dargestellt. Die Wiederherstellung des viralen Umschlags auf einer repräsentativen Virion ist in Abbildung 2B (blaue Isooberfläche) dargestellt.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51366/51366fig1highres.jpg" src="/files/ftp_upload/51366/51366fig1.jpg" style="opacity: 0.9; font-size: 14.399999618530273px; line-height: 28px;">
Abbildung 1. AFM-Bildgebung von Perlen und VSV auf der funktionalisierten PEGG-Oberfläche. AFM-Scan von 36 nm biotinylierten Perlen (A, B) und einem VSV Virion (C), der mit einem biotinylierten VSVG-Antikörper an der Oberfläche verankert ist. AFM wurde im AC-Luft-Topographie-Scan-Modus durchgeführt. Der Spitzenradius beträgt <25 nm und Messungen wurden unter Kraftmodulation und Lichtabstich durchgeführt. Die Spitze hatte eine Kraftkonstante von 3 N/m und Resonanzfrequenz 75 kHz mit einer Unsicherheit von 15 kHz. Während die Perlen während des AFM-Scans ihre Höhe beibehalten haben, hat das VSV Virion einen deutlich kleineren Jungmodul und ist in der Höhe deutlich verformt. In diesem Bild wurde das Virion speziell mit einem steifen Ausleger im Abstichmodus abgebildet, der eine kleine xy-Konvolution erzeugte (zur Bestimmung der Spitze vs. stumpfes Ende des Virus) und der Höhenunterschied zwischen der Spitze und dem stumpfen Ende des Virus wird verwendet, um zusätzliche Proteindichte am stumpfen Ende des Virus19zu erkennen.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51366/51366fig2highres.jpg" src="/files/ftp_upload/51366/51366fig2.jpg"> Abbildung 2. Fluoreszenzbasierte Abbildung von VSV-Virionen auf der funktionalisierten Oberfläche von PEGG. A) rekombinante VSV-Virionen, die auf der PEGG-Oberfläche mit einem biotinylierten Anti-VSVG-Antikörper in Weitfeldfluoreszenz immobilisiert wurden. B) Hochauflösende Fluoreszenzrekonstruktion der Anhüllung von VSV, die durch einen biotinylierten Anti-VSVG-Antikörper an der PEGG-Oberfläche befestigt ist und mit Alexa 647-beschrifteten Anti-VSVG-Antikörpern verziert ist (Methode 5A wurde zur Erstellung dieser Bilder verwendet). Die blaue Oberfläche ist eine 2D-Isooberflächenprojektion. Links zeigt ein Modell des kugelförmigen Virus. 
<table border="0" cellpadding="0" cellspacing="0" width="212"><colgroup>
<col>
<col>
</colgroup>
<tbody>
<tr height="27">
<td height="27" style="height:27px;width:119px;">Coverslip Größe</td>
			<td style="width:93px;">Flüssigkeit</td>
		</tr>
<tr height="20">
<td height="20" style="height:20px;">40 mm</td>
			<td>65 l</td>
		</tr>
<tr height="20">
<td height="20" style="height:20px;">35 mm</td>
			<td>50 l</td>
		</tr>
<tr height="20">
<td height="20" style="height:20px;">30 mm</td>
			<td>36 l</td>
		</tr>
<tr height="20">
<td height="20" style="height:20px;">25 mm</td>
			<td>25 l</td>
		</tr>
<tr height="20">
<td height="20" style="height:20px;">20 mm</td>
			<td>16 l</td>
		</tr>
</tbody></table>Tabelle 1. Fluid aliquots nach Deckslipgröße.

Watch this Scientific Journal Video about Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging at JoVE.com

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Die Befestigung von Virionen an einer Oberfläche ist eine Voraussetzung für eine einzelne Virionen-Bildgebung durch Super-Resolution Fluoreszenz-Bildgebung oder Atomkraftmikroskopie (AFM). Hier zeigen wir ein Probenvorbereitungsverfahren zur kontrollierten Haftung von Virionen auf Glasoberflächen, die für den Einsatz in AFM und Super-Auflösungfluoreszenz-Bildgebung geeignet sind.

Probenvorbereitung für Single Virion Atomic Force Microscopy und Super-Resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

sample-preparation-for-single-virion-atomic-force-microscopy-super

Research

JoVE Journal

Biology

9.4K Aufrufe.  University of Utah. Die Befestigung von Virionen an einer Oberfläche ist eine Voraussetzung für eine einzelne Virionen-Bildgebung durch Super-Resolution Fluoreszenz-Bildgebung oder Atomkraftmikroskopie (AFM). Hier zeigen wir ein Probenvorbereitungsverfahren zur kontrollierten Haftung von Virionen auf Glasoberflächen, die für den Einsatz in AFM und Super-Auflösungfluoreszenz-Bildgebung geeignet sind.

Serie: Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Zeitraffer-Imaging der Mitose Nach siRNA-Transfektion

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Forschung

Biologie

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

Super-Resolution Imaging der Bakterien Abteilung Machinery

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

Test Samples for Optimizing STORM Super-Resolution Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the 'mislocalization' phenomenon.

We describe the preparation of three test samples and how they can be used to optimize and assess the performance of STORM microscopes. Using these examples we show how to acquire raw data and then process it to acquire super-resolution images in cells of approximately 30-50 nm resolution.

Testproben zur Optimierung STORM Super Resolution Mikroskopie

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

Schnelle Analyse und Exploration von Fluoreszenzmikroskopie Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

Open-Source-Einzelpartikelanalyse für Super Resolution Mikroskopie mit VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

Grundzustand Erschöpfung Super-Resolution Imaging in Säugerzellen

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

Probenvorbereitung und Bildgebung von Exosomen von Transmissions-Elektronenmikroskopie

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

Miniaturisierte Probenvorbereitung für Transmissions-Elektronenmikroskopie

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

Biologische Probenvorbereitung durch Hochdruck gefrieren, Mikrowellen-gestützte Kontrastverstärkung und minimale Harz einbetten für Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

Hochauflösende Live-Cell-Bildgebung subzellulärer Strukturen

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...