Name: Kartierung der absoluten DNA-Dichte in Zellkernen mit Hilfe der Einzelmolekül-Lokalisierungsmikroskopie (Video) | JoVE Journal
Uploaded: 2025-11-11T14:00:00.000+00:00
Duration: 10 min 57 s
Description: Erfahren Sie, wie Sie absolute DNA-Dichten in Zellkernen mit Einzelmolekül-Lokalisierungsmikroskopie und Voronoi-Tessellation messen. Diese Methode ermöglicht präzise Analysen der Chromatinarchitektur in verschiedenen Zellzyklusphasen.

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<li>Ovesny, M., Krizek, P., Borkovec, J., Svindrych, Z., Hagen, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ThunderSTORM%3A+a+comprehensive+ImageJ+plug-in+for+PALM+and+STORM+data+analysis+and+super-resolution+imaging%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging.</a> Bioinformatics. 30 (16), 2389-2390 (2014).</li>
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<li>Strickfaden, H., Xu, Z. Z., Hendzel, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualization+of+miniSOG+tagged+DNA+repair+proteins+in+combination+with+Electron+Spectroscopic+Imaging+%28ESI%29%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Visualization of miniSOG tagged DNA repair proteins in combination with Electron Spectroscopic Imaging (ESI).</a> Journal of Visualized Experiments. (103), e52893(2015).</li>
<li>Xie, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BRD2+compartmentalizes+the+accessible+genome%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">BRD2 compartmentalizes the accessible genome.</a> Nature Genetics. 54 (4), 481-491 (2022).</li>
<li>Heo, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nuclear+softening+expedites+interstitial+cell+migration+in+fibrous+networks+and+dense+connective+tissues%5BTitle%5D)+AND+%22Science+Advances%22%5BJournal%5D)">Nuclear softening expedites interstitial cell migration in fibrous networks and dense connective tissues.</a> Science Advances. 6 (25), (2020).</li>
<li>Gelléri, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((True-to-scale+DNA-density+maps+correlate+with+major+accessibility+differences+between+active+and+inactive+chromatin%5BTitle%5D)+AND+%22bioRxiv%22%5BJournal%5D)">True-to-scale DNA-density maps correlate with major accessibility differences between active and inactive chromatin.</a> bioRxiv. , (2022).</li>
<li>Derenzini, M., Olins, A. L., Olins, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatin+structure+in+situ%3A+the+contribution+of+DNA+ultrastructural+cytochemistry%5BTitle%5D)+AND+%22European+Journal+of+Histochemistry%22%5BJournal%5D)">Chromatin structure in situ: the contribution of DNA ultrastructural cytochemistry.</a> European Journal of Histochemistry. 58 (1), 2307(2014).</li>
<li>Cremer, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+4D+nucleome%3A+Evidence+for+a+dynamic+nuclear+landscape+based+on+co-aligned+active+and+inactive+nuclear+compartments%5BTitle%5D)+AND+%22FEBS+Letters%22%5BJournal%5D)">The 4D nucleome: Evidence for a dynamic nuclear landscape based on co-aligned active and inactive nuclear compartments.</a> FEBS Letters. 589, 2931-2943 (2015).</li>
<li>Shah, F. A., Johansson, B. R., Thomsen, P., Palmquist, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrastructural+evaluation+of+shrinkage+artefacts+induced+by+fixatives+and+embedding+resins+on+osteocyte+processes+and+pericellular+space+dimensions%5BTitle%5D)+AND+%22Journal+of+Biomedical+Materials+Research+A%22%5BJournal%5D)">Ultrastructural evaluation of shrinkage artefacts induced by fixatives and embedding resins on osteocyte processes and pericellular space dimensions.</a> Journal of Biomedical Materials Research A. 103 (4), 1565-1576 (2015).</li>
<li>Gavelis, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dinoflagellate+nucleus+contains+an+extensive+endomembrane+network%2C+the+nuclear+net%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Dinoflagellate nucleus contains an extensive endomembrane network, the nuclear net.</a> Scientific Reports. 9 (1), 839(2019).</li>
<li>Rouquette, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Revealing+the+high-resolution+three-dimensional+network+of+chromatin+and+interchromatin+space%3A+a+novel+electron-microscopic+approach+to+reconstructing+nuclear+architecture%5BTitle%5D)+AND+%22Chromosome+Research%22%5BJournal%5D)">Revealing the high-resolution three-dimensional network of chromatin and interchromatin space: a novel electron-microscopic approach to reconstructing nuclear architecture.</a> Chromosome Research. 17 (6), 801-810 (2009).</li>
<li>Gorisch, S. M., Richter, K., Scheuermann, M. O., Herrmann, H., Lichter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diffusion-limited+compartmentalization+of+mammalian+cell+nuclei+assessed+by+microinjected+macromolecules%5BTitle%5D)+AND+%22Experimental+Cell+Research%22%5BJournal%5D)">Diffusion-limited compartmentalization of mammalian cell nuclei assessed by microinjected macromolecules.</a> Experimental Cell Research. 289 (2), 282-294 (2003).</li>
<li>Lenart, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nuclear+envelope+breakdown+in+starfish+oocytes+proceeds+by+partial+NPC+disassembly+followed+by+a+rapidly+spreading+fenestration+of+nuclear+membranes%5BTitle%5D)+AND+%22The+Journal+of+Cell+Biology%22%5BJournal%5D)">Nuclear envelope breakdown in starfish oocytes proceeds by partial NPC disassembly followed by a rapidly spreading fenestration of nuclear membranes.</a> The Journal of Cell Biology. 160 (7), 1055-1068 (2003).</li>
<li>Verschure, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Condensed+chromatin+domains+in+the+mammalian+nucleus+are+accessible+to+large+macromolecules%5BTitle%5D)+AND+%22EMBO+Reports%22%5BJournal%5D)">Condensed chromatin domains in the mammalian nucleus are accessible to large macromolecules.</a> EMBO Reports. 4 (9), 861-866 (2003).</li>
<li>Strickfaden, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Condensed+chromatin+behaves+like+a+solid+on+the+mesoscale+in+vitro+and+in+living+cells%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Condensed chromatin behaves like a solid on the mesoscale in vitro and in living cells.</a> Cell. 183 (7), 1772-1784 (2020).</li>
<li>Lin, Y. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((M33+condenses+chromatin+through+nuclear+body+formation+and+methylation+of+both+histone+H3+lysine+9+and+lysine+27%5BTitle%5D)+AND+%22Biochimica+et+Biophysica+Acta.+Molecular+Cell+Research%22%5BJournal%5D)">M33 condenses chromatin through nuclear body formation and methylation of both histone H3 lysine 9 and lysine 27.</a> Biochimica et Biophysica Acta. Molecular Cell Research. 1868 (11), 119100(2021).</li>
<li>Strickfaden, H., Sharma, A. K., Hendzel, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+charge-dependent+phase+transition+determines+interphase+chromatin+organization%5BTitle%5D)+AND+%22bioRxiv%22%5BJournal%5D)">A charge-dependent phase transition determines interphase chromatin organization.</a> bioRxiv. , (2019).</li>
</ol>

<table><tbody><tr><td><stark>Zellkultur</stark></td><td></td><td></td><td></td></tr><tr><td>&Micro;-Schüssel 35 mm, hoher Gitter-500 Glasboden</td><td>Ibidi</td><td>81168</td><td></td></tr><tr><td>C3H 10T1/2</td><td>IMB (Niehrs Lab)</td><td></td><td></td></tr><tr><td>DMEM</td><td>ThermoFisher</td><td>12320032</td><td></td></tr><tr><td>dPBS</td><td>ThermoFisher</td><td>14190144</td><td></td></tr><tr><td>FBS</td><td>Lebenstechnologien</td><td>16000-044</td><td></td></tr><tr><td>Hela</td><td>Mikroskopie-Kernanlage (IMB)</td><td></td><td></td></tr><tr><td>HFB</td><td>IMB (Kubben Lab)</td><td></td><td></td></tr><tr><td>L-Glutamin</td><td>Sigma-Aldrich</td><td>G7513</td><td></td></tr><tr><td>Nicht-essentielle Aminosäuren und Vitamine für HFB</td><td></td><td></td><td></td></tr><tr><td>Natriumpyruvat</td><td>S8636</td><td></td><td></td></tr><tr><td>Sample Prep</td><td></td><td></td><td></td></tr><tr><td>Katalase</td><td>Merck</td><td>2593710</td><td></td></tr><tr><td>Traubenzucker</td><td>ThermoFisher</td><td>241922500</td><td></td></tr><tr><td>Glukoseoxidase</td><td>Merck</td><td>49180</td><td></td></tr><tr><td>Paraformaldehyd</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>RNase-Cocktail</td><td>ThermoFisher</td><td>AM2286</td><td></td></tr><tr><td>SYTOX Orange</td><td>ThermoFisher</td><td>S11368</td><td></td></tr><tr><td>TetraSpeck Fluoreszierende Mikrosphären-Sampler-Kit</td><td>ThermoFisher</td><td>T7284</td><td></td></tr><tr><td>Triton X-100</td><td>ThermoFisher</td><td>327372500</td><td></td></tr><tr><td><stark>Software</stark></td><td></td><td></td><td></td></tr><tr><td>BioFormate</td><td>OpenMicroscopy.org</td><td></td><td>Open-Source-Software https://www.openmicroscopy.org/bio-formats/</td></tr><tr><td>CellProfiler v4.2.1</td><td>CellProfiler.org</td><td></td><td>Open-Source-Software https://cellprofiler.org</td></tr><tr><td>Fidschi</td><td>nih.gov</td><td></td><td>Open-Source-Software https://imagej.net/software/fiji/?Downloads</td></tr><tr><td>LAND</td><td>nih.gov</td><td></td><td>Open-Source-Software https://github.com/Jan-NM/LAND</td></tr><tr><td>MatLab 2021</td><td>Mathematische Werke</td><td></td><td>Kommerzielle Software – benötigt "Image Processing Toolbox"</td></tr><tr><td>R v.4.. 0.3</td><td>r-project.org</td><td></td><td>Open-Source-Software https://www.r-project.org</td></tr><tr><td>ThunderSTORM v1.3</td><td></td><td></td><td>Open-Source-Software https://zitmen.github.io/thunderstorm/</td></tr><tr><td><stark>Mikroskope: </stark></td><td></td><td></td><td></td></tr><tr><td>AF 7000</td><td>Leica</td><td></td><td></td></tr><tr><td>Leica-Schäferhund</td><td>Leica</td><td></td><td></td></tr><tr><td>SMLM-Mikroskop</td><td>Cremer-Labor</td><td></td><td>maßgeschneidert von Dr. S-Y. Chen</td></tr></tbody></table>

kartierung der absoluten dna-dichte in zellkernen mit hilfe der einzelmolekül-lokalisierungsmikroskopie

Mapping Absolute DNA Density in Cell Nuclei using Single-molecule Localization Microscopy

Kartierung der absoluten DNA-Dichte in Zellkernen mit Hilfe der Einzelmolekül-Lokalisierungsmikroskopie

Within the cell nucleus, silent genes are generally located in chromatin areas of high density called heterochromatin, whereas active genes can be mostly ...

mapping-absolute-dna-density-cell-nuclei-using-single-molecule

Research

Forschung

JoVE Journal

Biology

Biologie

Einzel-Molekül-Mikroskopie approch neue Einblicke in atomare Transport.

Single-Molecule Imaging of Nuclear Transport

Wir beschreiben eine robuste und vielseitige Protokoll zur Analyse von 3D-Kern Architektur DNA FISH mit direkt markierten fluoreszierenden Sonden.

Robust 3D DNA FISH Mit Direkt markierten Sonden

Photoaktivierte Lokalisationsmikroskopie (PALM), kombiniert mit Einzelmolekül-Tracking ermöglicht die direkte Beobachtung und Quantifizierung von DNA-Protein-Interaktionen in lebenden Escherichia coli-Zellen.

Visualisierung von Protein-DNA-Wechselwirkungen in der lebenden Bakterienzellen Verwendung Photoactivated Einzelmolekül-Tracking

Dargestellt ist eine flexible Informatik Workflow ermöglicht Multiplex bildbasierte Analyse fluoreszenzmarkierten Zellen. Der Workflow quantifiziert nukleäre und zytoplasmatische Marker und berechnet Marker Translokation zwischen diesen Fächern. Verfahren für Störung von Zellen mit siRNA und zuverlässige Methode zur Markerdetektion durch indirekte Immunfluoreszenz in 96-Well-Formaten zur Verfügung gestellt.

Workflow für Hoch Inhalt, einzelne Zell Quantifizierung der Fluoreszenzmarker von Universal Mikroskop Daten, Unterstützung von Open Source Software

Neu entdeckte oxidierte Formen von 5-Methylcytosine (Oxi-mCs), 5-Hydroxymethylcytosine (5hmC), 5-Formylcytosine (5-Fu) und 5-Carboxylcytosine (5caC) kann verschiedene DNA-Modifikationen mit einzigartigen funktionellen Rollen vertreten. Hier ist ein semi-quantitative Workflow zur Visualisierung der räumlichen Verteilung Oxi-mCs, Signal Intensität Profilierung und NS1 beschrieben.

Immunostaining für DNA-Modifikationen: computergestützte Analyse der konfokalen Bilder

Die 3D-Einzelmolekül-Lokalisationsmikroskopie wird verwendet, um die räumlichen Positionen und Bewegungsbahnen fluoreszierend markierter Proteine in lebenden Bakterienzellen zu untersuchen. Das hier beschriebene experimentelle und Datenanalyseprotokoll bestimmt das vorherrschende diffusive Verhalten von zytosolischen Proteinen auf der Grundlage gepoolter Einzelmolekülbahnen.

Single-Molecule Tracking-Mikroskopie - Ein Werkzeug zur Bestimmung der diffusiven Zustände von zytosolischen Molekülen

3D-Mehrfarben-DNA-FISH stellt ein Werkzeug dar, um mehrere genomische Loci innerhalb von 3D-konservierten Kernen zu visualisieren und ihre wechselseitigen Wechselwirkungen und Lokalisierungen innerhalb des Kernraums auf einer einzigen Zellebene eindeutig zu definieren. Hier wird ein Schritt-für-Schritt-Protokoll für ein breites Spektrum menschlicher Primärzellen beschrieben.

3D Multicolor DNA FISH Tool zur Erforschung der Kernarchitektur in menschlichen Primärzellen

Dieses Protokoll beschreibt die Single-Antibody-Markierung (SAL), um die nanoskalige räumliche Organisation von Plasmamembranproteinen zu klären. Durch die Nutzung kumulativer Antikörper-Epitope-Interaktionen auf Einzelmolekülebene kartiert Membran-SAL (mSAL) lokale Epitope-Verteilungen und erfasst gleichzeitig das Antikörperbindungsverhalten in der nativen zellulären Umgebung.

Einzelmolekül-Lokalisationsmikroskopie von Membranproteinen mittels Einzel-Antikörper-Markierung

Wir präsentieren ein dreifarbiges Chromatin-Einzelmolekül-Lokalisationsmikroskopie-(SMLM)-Färbungs- und Analyseverfahren, das eine reproduzierbare Abbildung von Euchromatin, Heterochromatin und RNAP II für die räumliche Analyse ermöglicht. Dieses Protokoll ermöglicht eine effiziente mehrfarbige Markierung in dichten nuklearen Umgebungen, einschließlich chromatinassoziierter Ziele, und ermöglicht eine zuverlässige gleichzeitige Erkennung.

Ein mehrschichtiges Einzelmolekül-Lokalisationsmikroskopieprotokoll zur Untersuchung von Chromatin in der dichten Kernumgebung

Dieses Protokoll bietet detaillierte Operationen zur Probenvorbereitung, Datenerhebung und Analyse von DNA-intrinsischen Fluoreszenzspektren.

Spektroskopische Superauflösungsbildgebung von DNA-Molekülen unter Verwendung von intrinsischem Kontrast

Dieses Protokoll bietet einen effizienten Arbeitsablauf für die mikroskopische Mikroskopieabbildung isolierter Arabidopsis-Kerne durch optimierte Isolierung, Fixierung und Markierung, um eine zuverlässige nanoskalige Visualisierung von Chromatin und RNA-Polymerase II zu erreichen. Dieser Ansatz kann leicht an andere chromatinassoziierte Proteine oder Histonmodifikationen für hochauflösende Bildgebung angepasst werden.

Ein stromlinienförmiges Protokoll für Einzelmolekül-Lokalisationsmikroskopie in Arabidopsis-Kernen

Erfahren Sie, wie Sie absolute DNA-Dichten in Zellkernen mit Einzelmolekül-Lokalisierungsmikroskopie und Voronoi-Tessellation messen. Diese Methode ermöglicht präzise Analysen der Chromatinarchitektur in verschiedenen Zellzyklusphasen.

Kartierung der absoluten DNA-Dichte in Zellkernen mit Hilfe der Einzelmolekül-Lokalisierungsmikroskopie

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