Wir danken Harald Hess und Eric Betzig für den Zugang zum PALM Mikroskop für proof-of-principle Experimente, Richard Fetter für den Austausch Fixierung Protokollen, Reagenzien und Ermutigung. Wir danken Michael Davidson, Geraldine Seydoux, Stefan Eimer, Rudolf Leube, Keith Nehrke, Christian Frøkjr-Jensen, Aude Ada-Nguema und Marc Hammarlund für die DNA-Konstrukte. Wir danken auch Carl Zeiss Inc. für den Zugang zu den Zeiss PAL-M, eine Beta-Version des Zeiss ELYRA P.1 PALM Mikroskop.

<ol>
	<li>Polishchuk, R. 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=Correlative+light-electron+microscopy+reveals+the+tubular-saccular+ultrastructure+of+carriers+operating+between+Golgi+apparatus+and+plasma+membrane.">Correlative light-electron microscopy reveals the tubular-saccular ultrastructure of carriers operating between Golgi apparatus and plasma membrane.</a> J. Cell Biol. 148, 45-58 (2000).</li><li>Oberti, D., Kirschmann, M. A., Hahnloser, R. H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlative+microscopy+of+densely+labeled+projection+neurons+using+neural+tracers.">Correlative microscopy of densely labeled projection neurons using neural tracers.</a> Front. Neuroanat. 4, 24 (2010).</li><li>Bishop, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+branding+efficiently+correlates+light+and+electron+microscopy.">Near-infrared branding efficiently correlates light and electron microscopy.</a> Nat. Meth. 8, 568-570 (2011).</li><li>Sims, P. A., Hardin, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-integrated+transmission+electron+microscopy+images:+integrating+fluorescence+microscopy+with+transmission+electron+microscopy.">Fluorescence-integrated transmission electron microscopy images: integrating fluorescence microscopy with transmission electron microscopy.</a> Methods Mol. Biol. 369, 291-308 (2007).</li><li>Micheva, K., Smith, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Array+tomography:+a+new+tool+for+imaging+the+molecular+architecture+and+ultrastructure+of+neural+circuits.">Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits.</a> Neuron. 55, 25-36 (2007).</li><li>Nixon, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+method+for+cryofixation+and+correlative+light,+electron+microscopy+and+tomography+of+zebrafish+embryos.">A single method for cryofixation and correlative light, electron microscopy and tomography of zebrafish embryos.</a> Traffic. 10, 131-136 (2009).</li><li>Kukulski, W., 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=Correlated+fluorescence+and+3D+electron+microscopy+with+high+sensitivity+and+spatial+precision.">Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision.</a> J. Cell Biol. 192, 111-119 (2011).</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>Rust, M. J., Bates, M., Zhuang, X. <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+(STORM).">Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).</a> Nat. Methods. 3, 793-795 (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>Weibull, C., Christiansson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+proteins+and+membrane+lipids+during+low+temperature+embedding+of+biological+material+for+electron+microscopy.">Extraction of proteins and membrane lipids during low temperature embedding of biological material for electron microscopy.</a> J. Microsc. 142, 79-86 (1986).</li><li>Watanabe, 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=Protein+localization+in+electron+micrographs+using+fluorescence+nanoscopy.">Protein localization in electron micrographs using fluorescence nanoscopy.</a> Nat. Methods. 8, 80-84 (2011).</li><li>F lling, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+nanoscopy+by+ground-state+depletion+and+single-molecule+return.">Fluorescence nanoscopy by ground-state depletion and single-molecule return.</a> Nat. Methods. 5, 943-945 (2008).</li><li>Gustafsson, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surpassing+the+lateral+resolution+limit+by+a+factor+of+two+using+structured+illumination+microscopy.">Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy.</a> J. Microsc. 198, 82-87 (2000).</li><li>Wombacher, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+super-resolution+imaging+with+trimethoprim+conjugates.">Live-cell super-resolution imaging with trimethoprim conjugates.</a> Nature Methods. 7, 717-719 (2010).</li><li>Dertinger, T., Colyer, R., Iyer, G., Weiss, S., Enderlein, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast,+background-free,+3D+super-resolution+optical+fluctuation+imaging+(SOFI).">Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI).</a> PNAS. 106, 22287-22292 (2009).</li><li>Burnette, D. T., Sengupta, P., Dai, Y., Lippincott-Schwartz, J., Kachar, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bleaching/blinking+assisted+localization+microscopy+for+superresolution+imaging+using+standard+fluorescent+molecules.">Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules.</a> PNAS. , (2011).</li><li>Annibale, P., Vanni, S., Scarselli, M., Rothlisberger, U., Radenovic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+clustering+artifacts+in+photoactivated+localization+microscopy.">Identification of clustering artifacts in photoactivated localization microscopy.</a> Nat. Meth. 8, 527-528 (2011).</li></ol>

Produktion und den freien Zugang zu diesem Artikel wird von Carl Zeiss, Inc. gesponsert

Hier beschreiben wir, wie man fluoreszierende Proteine ​​erhalten in Kunststoff, lokalisieren die fluoreszierenden Proteine ​​in den Abschnitten und Bild die Ultrastruktur mittels Elektronenmikroskopie. Die Proteine ​​wurden unterhalb der Beugungsgrenze mit PALM Mikroskopie Nanometer-Auflösung lokalisiert. Um dieses Protokoll zu bestimmten Proben anzupassen, sollte die folgenden Parameter berücksichtigt werden: Fluorophor, Quantifizierung und Ausrichtung. Die Wahl des fluoreszierenden Proteins oder organischen Fluorophor hängt von der Anwendung und dem Modellsystem. Wir haben eine Vielzahl von fluoreszierenden Proteinen, einschließlich EGFP, YFP, Citrin, mEosFP, mEos2, tdEos, Morange, PA-mCherry und Dendra 12 getestet. Die Erhaltung der Fluoreszenz von jedem Fluorophor war ähnlich, was darauf hindeutet, dass alle fluoreszierenden Proteinen erhalten kann mit dem beschriebenen Verfahren werden. Wir wählten tdEos weil es ausgesprochen gut in C. elegans, blieb Proteine ​​funktionell, wenn die tdEos verschmolzen, und weil seinePhotoaktivierung Eigenschaften waren optimal für PALM Mikroskopie. Allerdings hat Aggregation oder nicht Ausdruck tdEos wurde gelegentlich beobachtet 12. Je nach Anwendung kann ein anderes Fluorophor besser geeignet sein. In vielen Fällen ist es nicht notwendig, einen photoaktivierten fluoreszierenden Proteins zu verwenden. Einfache korrelative Fluoreszenz Elektronenmikroskopie erfordert keine photoaktivierten Fluoreszenzprotein. GFP oder organische Farbstoffe zur Bilder Fluoreszenz markierte Proteine ​​in den Abschnitten oberhalb der Beugungsgrenze verwendet werden. Beispielsweise kann ein Bild, ein Axon in einem Neuropil mittels Fluoreszenzmikroskopie und korrelieren das Fluoreszenzsignal einer bestimmten Axons in einer elektronenmikroskopischen Aufnahme durch Abbilden der Fluoreszenz an einem Fluoreszenzmikroskop. Andere Superauflösung Techniken wie stimulierte Emission Mikroskopie (STED) 12, Grundzustand Depletion-Mikroskopie von einzelnen Moleküls Rücklauf (GSDIM) 13 gefolgt, undstrukturierten Beleuchtung Mikroskopie (SIM) 14, erfordern keine photoaktivierten fluoreszierende Proteine. Darüber hinaus sind super-resolution imaging Techniken, organischen Farbstoffen 9,15,16 oder die intrinsische Eigenschaft von fluoreszierenden Sonden 17 verwenden leicht anwendbar. In PALM, kann die Anzahl der Moleküle, da quantifizierte Fluoreszenz jedes Molekül räumlich und zeitlich getrennt werden. Allerdings kann die Quantifizierung für vier Gründen irreführend: Oxidation, Unterschätzung, Überzählen und Überexpression. Erstens kann ein Bruchteil der fluoreszierenden Proteine ​​denaturiert oder oxidiert während Probenprozessierungsvorrichtung 5,12. Obwohl ~ 90% des Fluoreszenzsignals durch Fixierung und Einbettung in unserem Protokoll erhalten wurde, kann eine Oxidation des fluoreszierenden Proteins auftreten, nachdem die Probe wurde geschnitten und die Oberfläche Sauerstoff ausgesetzt. Zweitens ist die Aktivierung der photoaktivierbaren Proteine ​​stochastischen und somit mehrere Moleküle könnenaktiviert in einer gegebenen beugungsbegrenzten Fleck 8. Fluoreszenz von den mehreren Moleküle wird als einer Stelle erscheinen und somit die Gesamtzahl der Proteine ​​wird undercounted werden. Drittens kann ein ähnliches Problem zu Überzählen führen. In PALM wird jedes fluoreszierende Protein lokalisiert und dann auf &quot;Löschen&quot; durch Bleichen. Allerdings können fluoreszierende Proteine ​​aus dem dunklen Zustand, ohne dauerhaft gebleichte 18 zurückzukehren. Solche Moleküle werden dann mehrfach gezählt werden. Viertens tagged Proteine ​​als Transgene exprimiert und sind oft in mehreren Kopien, die zur Überexpression führen kann präsentieren. Daher kann die Quantifizierung von PALM für die Schätzung jedoch nicht genau bestimmen die Anzahl von Molekülen in einer bestimmten Position ist. Die Ausrichtung eines PALM Bild mit einer elektronenmikroskopischen Aufnahme kann auch wegen der Auflösung Unterschied in Licht und Elektronenmikroskopie und Verzerrung, die durch den Elektronenstrahl hervorgerufen Herausforderung sein. Goldpartikel dienen als dicht locsierte Bezugsmarkierungen in elektronenmikroskopischen Aufnahmen. Allerdings ist fluoresecence aus Gold Partikel nicht photoaktivierten und erscheint als eine große beugungsbegrenzten Fleck. Somit ist die Platzierung eines Fluoreszenzbilds auf einer elektronenmikroskopischen Aufnahme eine Schätzung. Verzerrungen können auch aus Wechselwirkungen der Elektronen mit dem Kunststoffteil entstehen. Acrylharze wie GMA sind weniger stabil unter dem Elektronenstrahl, und die Abmessungen des Kunststoffes verändert werden kann. Unter diesen Umständen kann das Ausrichten des Fluoreszenz mit Ultrastruktur die nicht-lineare Transformation der Bezugsmarkierungen.

<table border="1"><tbody><tr><td> Name des Reagens oder Ausrüstung </td><td> Firma </td><td> Katalognummer </td><td> Kommentare </td></tr><tr><td> Hochdruck-Gefrierschrank </td><td> ABRA </td><td> HPM 010 </td><td> Empact und HPM 100 von Leica oder hpf-01 ab Wohlwend können ebenfalls verwendet werden. </td></tr><tr><td> Automatisierte freeze Substitutionseinheit </td><td> Leica </td><td> AFS 2 </td><td> AFS 1 kann auch verwendet werden. </td></tr><tr><td> Zeiss PALM </td><td> Zeiss </td><td> ELYRA P.1 </td><td> Nikon und Vutara verkaufen auch Gewerbe PALM Mikroskope. </td></tr><tr><td> Rasterelektronenmikroskop </td><td> FEI </td><td> Nova nano </td><td> Andere hochauflösenden SEM Mikroskope verwendet werden kann. </td></tr><tr><td> Aceton </td><td> EMS </td><td> RT10016 </td><td></td></tr><tr><td> Ethanol </td><td> Sigma-Aldrich </td><td> 459.844-1L </td><td></td></tr><tr><td> Osmium tetroxide </td><td> EMS </td><td> RT19134 </td><td></td></tr><tr><td> Kaliumpermanganat </td><td> EMS </td><td> RT20200 </td><td></td></tr><tr><td> Albumin aus Rinderserum </td><td> Sigma-Aldrich </td><td> A3059-50G </td><td></td></tr><tr><td> Uranylacetat </td><td> Polysciences </td><td> 21447-25 </td><td> pH-Wert von Uranylacetat von diesem Unternehmen etwas höher ist. </td></tr><tr><td> Glycol-Methacrylat (GMA) </td><td> SPI </td><td> 02.630-AA </td><td> Low Säure, TEM Klasse. </td></tr><tr><td> N, N-Dimethyl-p-toluidin </td><td> Sigma-Aldrich </td><td> D9912 </td><td></td></tr><tr><td> Cryoröhrchen </td><td> Nalgene </td><td> 5000-0020 </td><td></td></tr><tr><td> Glasfläschchen </td><td> EMS </td><td> 72632 </td><td></td></tr><tr><td> Aclar Film </td><td> EMS </td><td> 50425-10 </td><td></td></tr><tr><td> BEEM Kapsel </td><td> EBSciences </td><td> TC </td><td> Polypropylen </td></tr><tr><td> 3/8 &quot;DISC Schläge </td><td> Ted Pella </td><td> 54741 </td><td></td></tr><tr><td> Gold-Nanopartikel </td><td> Mikrosphären-nanospheres.com </td><td> 790122-010 </td><td> Fordern 2x konzentrierte Lösung </td></tr></tbody></table>

nano-fem: protein localization using photo-activated localization microscopy and electron microscopy

Abbilden der Verteilung von Proteinen ist wesentlich für das Verständnis der Funktion von Proteinen in einer Zelle. Fluoreszenzmikroskopie wird weitgehend für Proteinlokalisierung, aber subzelluläre Zusammenhang ist oft in Fluoreszenz-Aufnahmen fehlen. Immuno-Elektronenmikroskopie, auf der anderen Seite kann lokalisieren Proteine, aber die Technik ist durch das Fehlen von Antikörpern kompatibel, schlechte Konservierung von Morphologie und begrenzt, da die meisten Antigene nicht zur Probenoberfläche ausgesetzt sind. Korrelativen Ansätzen kann die Fluoreszenz Bild aus einer ganzen Zelle erst einmal erwerben, entweder von Immun-Fluoreszenz oder genetisch markierten Proteine. Die Probe wird dann fixiert und für die Elektronenmikroskopie eingebettet und die Bilder korreliert werden 1-3. Jedoch schließt die niedriger Auflösung Fluoreszenzbild und das Fehlen von Bezugsmarkierungen die genaue Lokalisierung von Proteinen. Alternativ können Fluoreszenz-Bildgebung nach Erhalt der Probe in Kunststoff ausgeführt werden. Indieser Ansatz ist der Block geschnitten und Fluoreszenzbilder und elektronenmikroskopische Aufnahmen von dem gleichen Abschnitt sind 4-7 korreliert. Allerdings überdeckt die Beugungsgrenze des Lichts in dem korrelierten Bild die Positionen der einzelnen Moleküle, und die Fluoreszenz erstreckt sich oft über die Grenze der Zelle. Nano-Auflösung Fluoreszenz-Elektronenmikroskopie (nano-FEM), zur Lokalisierung von Proteinen im nanoskaligen Maßstab durch bildgebende den gleichen Abschnitten mit Foto-activated localization microscopy (PALM) und Elektronenmikroskopie. PALM überwindet die Beugungsgrenze durch bildgebende einzelner fluoreszierender Proteine ​​und anschließend Kartierung der Schwerpunkt jedes fluoreszierenden Fleck 8-10. Wir skizzieren die Nano-FEM-Technik in fünf Schritten. Zuerst wird die Probe fixiert und eingebettet unter Verwendung von Bedingungen, die die Fluoreszenz des markierten Proteinen zu erhalten. Zweitens sind die Harz-Blöcke in ultradünnen Segmente (70-80 nm), die montiert sind geschnitteneauf einem Deckglas. Drittens wird die Fluoreszenz in diesen Abschnitten mit dem Zeiss PALM Mikroskop abgebildet. Viertens werden Elektronen dichte Strukturen in den gleichen Abschnitten mit einem Rasterelektronenmikroskop abgebildet wird. Fünftens werden die Fluoreszenz-und elektronenmikroskopische Aufnahmen ausgerichtet mit Goldpartikeln Bezugsmarken. Zusammenfassend kann die subzelluläre Lokalisation von fluoreszent markierte Proteine ​​in einer Auflösung im Nanometerbereich auf etwa eine Woche bestimmt werden.

Watch this Scientific Journal Video about Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy at JoVE.com

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Wir beschreiben ein Verfahren zur Lokalisierung fluoreszent markierte Proteine ​​in elektronenmikroskopischen Aufnahmen. Fluoreszenz wird zuerst lokalisiert durch Foto-activated localization microscopy auf ultradünne Schnitte. Diese Bilder werden dann an elektronenmikroskopischen Aufnahmen von dem gleichen Abschnitt ausgerichtet ist.

Nano-FEM: Protein Localization Mit Photo-aktivierten Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

nano-fem-protein-localization-using-photo-activated-localization

Research

JoVE Journal

Biology

15.9K Aufrufe.  University of Utah. Wir beschreiben ein Verfahren zur Lokalisierung fluoreszent markierte Proteine ​​in elektronenmikroskopischen Aufnahmen. Fluoreszenz wird zuerst lokalisiert durch Foto-activated localization microscopy auf ultradünne Schnitte. Diese Bilder werden dann an elektronenmikroskopischen Aufnahmen von dem gleichen Abschnitt ausgerichtet ist.

Serie: Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Simultane Multicolor-Imaging biologischer Strukturen mit Fluoreszenzphotoaktivierung Lokalisationsmikroskopie

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Forschung

Biologie

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

Die Quantifizierung der Proteinexpression und Co-Lokalisation Mit Multiplexed immunhistochemischen Anfärbung und Multispektraltechnik

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and &#62; 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical microscopy.

Dreidimensionale Super Resolution Mikroskopie von F-Aktin durch interferometrische fotoaktivierte Lokalisationsmikroskopie (iPalm)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with ~15&#8211;27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of self-labeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondrial compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.

Here, we present a protocol for multi-color localization of single membrane proteins in organelles of live cells. To attach fluorophores, self-labeling proteins are used. Proteins, located in different membranes compartments of the same organelle, can be localized with a precision of ~18 nm.

Multi-Color-Lokalisierung Mikroskopie des einzigen Membranproteine in Organellen der lebenden Säugetier-Zellen

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a ...

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 ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

Visualisierung der Lymphknotenstruktur und Zelllokalisierung mittels Ex-Vivo Konfokalmikroskopie

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure.

Die CryoAPEX-Methode zur Elektronenmikroskopie-Analyse der Membranproteinlokalisierung in ultrastrukturell konservierten Zellen

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the &#945;-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

Analyzing the &amp;#945;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

Analyse des α-Actinin-Netzwerks in humanen iPSC-abgeleiteten Kardiomyozyten mittels Einzelmolekül-Lokalisierungsmikroskopie

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these &#34;time-resolved&#34; studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.

The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.

Kryo-elektronenmikroskopische Gittervorbereitung für zeitaufgelöste Studien mit einem neuartigen Robotersystem, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental ...

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.

The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.