Name: Mappatura della densità assoluta del DNA nei nuclei cellulari mediante microscopia a localizzazione a singola molecola (Video) | JoVE Journal
Uploaded: 2025-11-11T14:00:00.000+00:00
Duration: 10 min 57 s
Description: Scopri come misurare la densità assoluta del DNA nei nuclei cellulari aderenti utilizzando la tessellazione di Voronoi con microscopia SMLM. Questo metodo computazionale consente di analizzare l'architettura della cromatina con precisione nanometrica.

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</ol>

<table><tbody><tr><td><forte>Coltura cellulare</forte></td><td></td><td></td><td></td></tr><tr><td>&micro;-Dish 35 mm, alto Grid-500 Fondo in vetro</td><td>Ibidi</td><td>81168</td><td></td></tr><tr><td>C3H 10T1/2</td><td>IMB (Laboratorio Niehrs)</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>Life Technologies</td><td>16000-044</td><td></td></tr><tr><td>Hela</td><td>Struttura Centrale di Microscopia (IMB)</td><td></td><td></td></tr><tr><td>HFB</td><td>IMB (Laboratorio Kubben)</td><td></td><td></td></tr><tr><td>L-Glutammina</td><td>Sigma-Aldrich</td><td>G7513</td><td></td></tr><tr><td>Amminoacidi e vitamine non essenziali per l'HFB</td><td></td><td></td><td></td></tr><tr><td>Piruvato di sodio</td><td>S8636</td><td></td><td></td></tr><tr><td><forte>Preparazione del campione</forte></td><td></td><td></td><td></td></tr><tr><td>Catalasi</td><td>Merck</td><td>2593710</td><td></td></tr><tr><td>Glucosio</td><td>ThermoFisher</td><td>241922500</td><td></td></tr><tr><td>Glucosio ossidasi</td><td>Merck</td><td>49180</td><td></td></tr><tr><td>Paraformaldeide</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>Kit campionatore per microsfere fluorescenti TetraSpeck</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><forte>Software</forte></td><td></td><td></td><td></td></tr><tr><td>BioFormati</td><td>OpenMicroscopy.org</td><td></td><td>Software open source https://www.openmicroscopy.org/bio-formats/</td></tr><tr><td>CellProfiler v4.2.1</td><td>CellProfiler.org</td><td></td><td>software open source https://cellprofiler.org</td></tr><tr><td>Figi</td><td>nih.gov</td><td></td><td>software open source https://imagej.net/software/fiji/?Downloads</td></tr><tr><td>TERRA</td><td>nih.gov</td><td></td><td>Software open source https://github.com/Jan-NM/LAND</td></tr><tr><td>MatLab 2021</td><td>Matematica Works</td><td></td><td>software commerciale - richiede "Image Processing Toolbox"</td></tr><tr><td>R v.4.. 0.3</td><td>r-project.org</td><td></td><td>Software open source https://www.r-project.org</td></tr><tr><td>ThunderSTORM v1.3</td><td></td><td></td><td>Software open source https://zitmen.github.io/thunderstorm/</td></tr><tr><td><forte>Microscopi: </forte></td><td></td><td></td><td></td></tr><tr><td>AF 7000</td><td>Leica</td><td></td><td></td></tr><tr><td>Leica GSD</td><td>Leica</td><td></td><td></td></tr><tr><td>Microscopio SMLM</td><td>Laboratorio Cremer</td><td></td><td>costruita su misura dal dottor S-Y. Chen</td></tr></tbody></table>

mappatura della densità assoluta del dna nei nuclei cellulari mediante microscopia a localizzazione a singola molecola

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

Mappatura della densità assoluta del DNA nei nuclei cellulari mediante microscopia a localizzazione a singola molecola

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

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Recentemente scoperto forme ossidate di 5-metilcitosina (oxi-mCs), 5-idrossimetilcitosina (5hmC), 5-formylcytosine (5fC) e 5-carboxylcytosine (5caC) può rappresentare distinte modificazioni del DNA con ruoli funzionali unici. Qui è descritto un flusso di lavoro semi-quantitativa per visualizzazione della distribuzione spaziale di oxi-mCs', profilatura di intensità di segnale e colocalizzazione.

Immunostaining per modificazioni del DNA: analisi computazionale delle immagini Confocal

La microscopia di localizzazione 3D a molecola singola viene utilizzata per sondare le posizioni spaziali e le traiettorie di movimento delle proteine fluorescenti etichettate nelle cellule batteriche viventi. Il protocollo sperimentale e di analisi dei dati descritto nel presente documento determina i comportamenti diffusivi prevalenti delle proteine citosoliche basate su traiettorie a singola molecola raggruppate.

Microscopia di tracciamento a singola molecola - Uno strumento per determinare gli stati diffusivi delle molecole citosoliche

IL DNA FISH multicolore 3D rappresenta uno strumento per visualizzare più loci genomici all'interno dei nuclei 3D conservati, definendo inequivocabilmente le loro interazioni reciproche e la localizzazione all'interno dello spazio nucleare a livello di singola cellula. Qui, un protocollo passo dopo passo è descritto per un ampio spettro di cellule primarie umane.

3D Multicolor DNA FISH Tool per studiare l'architettura nucleare nelle cellule primarie umane

Questo protocollo descrive la marcatura a singolo anticorpo (SAL) per risolvere l'organizzazione spaziale su scala nanometrica delle proteine della membrana plasmatica. Sfruttando le interazioni cumulative anticorpo-epitopo a livello di singola molecola, il SAL di membrana (mSAL) mappa le distribuzioni locali degli epitopi catturando contemporaneamente il comportamento di legame degli anticorpi nell'ambiente cellulare nativo.

Microscopia di localizzazione a singola molecola delle proteine di membrana utilizzando la marcatura a singolo anticorpo

Presentiamo un protocollo di colorazione e analisi per la microscopia di localizzazione a singola molecola della cromatina a tre colori (SMLM) che consente la mappatura riproducibile di eucromatina, eterocromatina e RNAP II per l'analisi spaziale. Questo protocollo consente un'efficiente marcatura multicolore in ambienti nucleari densi, inclusi bersagli associati alla cromatina, consentendo una rilevazione simultanea affidabile.

Un protocollo di microscopia a localizzazione di molecole singole multimarcatore per l'indagine della cromatina nell'ambiente nucleare denso

Questo protocollo fornisce operazioni dettagliate sulla preparazione del campione, raccolta dati e analisi degli spettri di fluorescenza intrinseca del DNA.

Imaging spettroscopico a super-risoluzione delle molecole di DNA utilizzando contrasto intrinseco

Questo protocollo presenta un flusso di lavoro efficiente per l'imaging microscopico di localizzazione a singola molecola di nuclei isolati di Arabidopsis, utilizzando isolamento, fissazione e marcatura ottimizzati per ottenere una visualizzazione affidabile su scala nanometrica della cromatina e della RNA Polimerasi II. Questo approccio può essere facilmente adattato ad altre proteine associate alla cromatina o modifiche agli istoni per immagini ad alta risoluzione.

Un protocollo semplificato per la microscopia di localizzazione a singola molecola nei nuclei di Arabidopsis

Scopri come misurare la densità assoluta del DNA nei nuclei cellulari aderenti utilizzando la tessellazione di Voronoi con microscopia SMLM. Questo metodo computazionale consente di analizzare l'architettura della cromatina con precisione nanometrica.

Mappatura della densità assoluta del DNA nei nuclei cellulari mediante microscopia a localizzazione a singola molecola

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