Name: Mapeando a densidade absoluta de DNA em núcleos celulares usando microscopia de localização de molécula única (Vídeo) | JoVE Journal
Uploaded: 2025-11-11T14:00:00.000+00:00
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Description: Aprenda a medir densidades absolutas de DNA em núcleos celulares usando tesselação de Voronoi e microscopia de localização de molécula única. Este método computacional permite investigar restrições espaciais na arquitetura da cromatina.

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</ol>

<table><tbody><tr><td><forte>Cultura Celular</forte></td><td></td><td></td><td></td></tr><tr><td>&micro;-Prato 35 mm, fundo de vidro Grid-500 alto</td><td>Ibidi</td><td>81168</td><td></td></tr><tr><td>C3H 10T1/2</td><td>IMB (Laboratório 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>Tecnologias de Vida</td><td>16000-044</td><td></td></tr><tr><td>Hela</td><td>Instalação Núcleo de Microscopia (IMB)</td><td></td><td></td></tr><tr><td>HFB</td><td>IMB (Laboratório Kubben)</td><td></td><td></td></tr><tr><td>L-Glutamina</td><td>Sigma-Aldrich</td><td>G7513</td><td></td></tr><tr><td>Aminoácidos e vitaminas não essenciais para HFB</td><td></td><td></td><td></td></tr><tr><td>Piruvato de Sódio</td><td>S8636</td><td></td><td></td></tr><tr><td><forte>Preparação de Amostra</forte></td><td></td><td></td><td></td></tr><tr><td>Catalase</td><td>Merck</td><td>2593710</td><td></td></tr><tr><td>Glicose</td><td>ThermoFisher</td><td>241922500</td><td></td></tr><tr><td>Glucose Oxidase</td><td>Merck</td><td>49180</td><td></td></tr><tr><td>Paraformaldeído</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 Laranja</td><td>ThermoFisher</td><td>S11368</td><td></td></tr><tr><td>Kit de Amostragem de Microsferas Fluorescentes 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>BioFormatos</td><td>OpenMicroscopy.org</td><td></td><td>Software de código aberto https://www.openmicroscopy.org/bio-formats/</td></tr><tr><td>CellProfiler v4.2.1</td><td>CellProfiler.org</td><td></td><td>Software de código aberto https://cellprofiler.org</td></tr><tr><td>Fiji</td><td>nih.gov</td><td></td><td>Software de código aberto https://imagej.net/software/fiji/?Downloads</td></tr><tr><td>TERRA</td><td>nih.gov</td><td></td><td>Software de código aberto https://github.com/Jan-NM/LAND</td></tr><tr><td>MatLab 2021</td><td>Matemática Funciona</td><td></td><td>software comercial - requer "Caixa de Ferramentas de Processamento de Imagens"</td></tr><tr><td>R v.4.. 0.3</td><td>r-project.org</td><td></td><td>Software de código aberto https://www.r-project.org</td></tr><tr><td>ThunderSTORM v1.3</td><td></td><td></td><td>Software de código aberto https://zitmen.github.io/thunderstorm/</td></tr><tr><td><forte>Microscópios: </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>Microscópio SMLM</td><td>Laboratório Cremer</td><td></td><td>Construído sob medida pelo Dr. S-Y. Chen</td></tr></tbody></table>

mapeando a densidade absoluta de dna em núcleos celulares usando microscopia de localização de molécula única

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

Mapeando a densidade absoluta de DNA em núcleos celulares usando microscopia de localização de molécula única

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

JoVE Journal

Biology

Biologia

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Robust FISH DNA 3D utilizando sondas Diretamente rotuladas

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Apresentado é um informática fluxo de trabalho flexível permitindo uma análise baseada em imagem multiplexado de células marcadas com fluorescência. O fluxo de trabalho quantifica marcadores nucleares e citoplasmáticos e calcula marcador translocação entre esses compartimentos. Os procedimentos são fornecidos por perturbação das células utilizando metodologia de siRNA e fiável para a detecção do marcador através de imunofluorescência indirecta em formatos de 96 poços.

Fluxo de trabalho para de alta conteúdo, Quantificação de células individuais de marcadores fluorescentes de Microscópio Universal Data, apoiada pela Open Source Software

Recém descoberto formas oxidadas de 5-metilcitosina (oxi-mCs), 5-Hidroximetilcitosina (5hmC), 5-formylcytosine (5-fC) e 5-carboxylcytosine (5caC) pode representar modificações de DNA distintas com funções únicas funcionais. Aqui, um fluxo de trabalho semi-quantitativa para visualização da distribuição espacial de oxi-mCs, perfis de intensidade de sinal e colocalization é descrito.

Immunostaining para modificações do DNA: análise computacional de imagens Confocal

a microscopia da localização da único-molécula 3D é utilizada para sondar as posições espaciais e as trajetórias do movimento de proteínas cDNAs etiquetadas em pilhas bacterianas vivas. O protocolo de análise experimental e de dados descrito neste documento determina os comportamentos diffusivos prevalentes de proteínas citosómicas baseadas em trajetórias unimoléculas agrupadas.

Microscopia de seguimento da único-molécula-uma ferramenta para determinar os Estados diffusive de moléculas cytosolic

O DNA 3D multicolor FISH representa uma ferramenta para visualizar loci múltiplogenômico dentro de núcleos preservados em 3D, definindo inequivocamente suas interações recíprocas e localização dentro do espaço nuclear em um único nível celular. Aqui, um protocolo passo a passo é descrito para um amplo espectro de células primárias humanas.

Ferramenta de peixe de DNA Multicolor 3D para estudar arquitetura nuclear em células primárias humanas

Este protocolo descreve a marcação de anticorpo único (SAL) para resolver a organização espacial em nanoescala das proteínas da membrana plasmática. Ao aproveitar as interações cumulativas anticorpo-epítopo no nível de molécula única, o SAL de membrana (mSAL) mapeia distribuições locais de epítopos enquanto captura simultaneamente o comportamento de ligação de anticorpos no ambiente celular nativo.

Microscopia de Localização de Molécula Única de Proteínas de Membrana usando Marcagem de Único Anticorpo

Apresentamos um protocolo de microscopia de localização de molécula única em três cores (SMLM) que permite o mapeamento reprodutível de eucromatina, heterocromatina e RNAP II para análise espacial. Esse protocolo permite a marcação eficiente de multicor em ambientes nucleares densos, incluindo alvos associados à cromatina, possibilitando detecção simultânea e confiável.

Um Protocolo de Microscopia de Localização de Molécula Única Multirótulo para Investigação da Cromatina em Ambiente Nuclear Denso

Este protocolo fornece operações detalhadas sobre preparação de amostras, coleta de dados e análise dos espectros de fluorescência intrínseca do DNA.

Imagem Espectroscópica de Super-resolução de Moléculas de DNA usando Contraste Intrínseco

Este protocolo apresenta um fluxo de trabalho eficiente para microscopia de localização de molécula única em núcleos isolados de Arabidopsis, utilizando isolamento, fixação e marcagem otimizados para alcançar visualização confiável em nanoescala da cromatina e da RNA Polimerase II. Essa abordagem pode ser facilmente adaptada a outras proteínas associadas à cromatina ou modificações de histonas para imagens de alta resolução.

Um Protocolo Simplificado para Microscopia de Localização de Molécula Única em Núcleos de Arabidopsis

Aprenda a medir densidades absolutas de DNA em núcleos celulares usando tesselação de Voronoi e microscopia de localização de molécula única. Este método computacional permite investigar restrições espaciais na arquitetura da cromatina.

Mapeando a densidade absoluta de DNA em núcleos celulares usando microscopia de localização de molécula única

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