Name: Tek Molekül Lokalizasyon Mikroskobu Kullanılarak Hücre Çekirdeğinde Mutlak DNA Yoğunluğunun Haritalanması (Video) | JoVE Journal
Uploaded: 2025-11-11T14:00:00.000+00:00
Duration: 10 min 57 s
Description: Keşfedin, tek molekül lokalizasyon mikroskobu ve Voronoi tessellation kullanarak hücre çekirdeğinde mutlak DNA yoğunluğunu haritalama yöntemini. Bu yaklaşım, kromatin mimarisindeki uzamsal kısıtlamaları incelemeyi sağlar.

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</ol>

<table><tbody><tr><td><Güçlü>Hücre Kültürü</Güçlü></td><td></td><td></td><td></td></tr><tr><td>&micro;-Çanak 35 mm, yüksek ızgara-500 Cam taban</td><td>İbidi</td><td>81168</td><td></td></tr><tr><td>C3H 10T1/2</td><td>IMB (Niehrs Laboratuvarı)</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>Yaşam Teknolojileri</td><td>16000-044</td><td></td></tr><tr><td>Hela</td><td>Mikroskopi Çekirdek Tesisi (IMB)</td><td></td><td></td></tr><tr><td>HFB</td><td>IMB (Kubben Laboratuvarı)</td><td></td><td></td></tr><tr><td>L-Glutamin</td><td>Sigma-Aldrich</td><td>G7513</td><td></td></tr><tr><td>HFB için Temel Olmayan Aminoasitler ve Vitaminler</td><td></td><td></td><td></td></tr><tr><td>Sodyum Piruvat</td><td>S8636</td><td></td><td></td></tr><tr><td>Örnek Hazırlık</td><td></td><td></td><td></td></tr><tr><td>Katalaz</td><td>Merck</td><td>2593710</td><td></td></tr><tr><td>Glikoz</td><td>ThermoFisher</td><td>241922500</td><td></td></tr><tr><td>Glikoz Oksidaz</td><td>Merck</td><td>49180</td><td></td></tr><tr><td>Paraformaldehit</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>RNase Kokteyli</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 Floresan Mikrosferler Örnekleyici Kiti</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>Software</td><td></td><td></td><td></td></tr><tr><td>BioFormatlar</td><td>OpenMicroscopy.org</td><td></td><td>Açık kaynak yazılım https://www.openmicroscopy.org/bio-formats/</td></tr><tr><td>CellProfiler v4.2.1</td><td>CellProfiler.org</td><td></td><td>Açık kaynak yazılım https://cellprofiler.org</td></tr><tr><td>Fiji</td><td>nih.gov</td><td></td><td>Açık kaynak yazılım https://imagej.net/software/fiji/?Downloads</td></tr><tr><td>KARA</td><td>nih.gov</td><td></td><td>Açık kaynak yazılım https://github.com/Jan-NM/LAND</td></tr><tr><td>MatLab 2021</td><td>Matematik Eserleri</td><td></td><td>ticari yazılım - "Görüntü İşleme Araç Kutusu" gerektirir</td></tr><tr><td>R v.4.. 0.3</td><td>r-project.org</td><td></td><td>Açık kaynak yazılım https://www.r-project.org</td></tr><tr><td>ThunderSTORM v1.3</td><td></td><td></td><td>Açık kaynak yazılım https://zitmen.github.io/thunderstorm/</td></tr><tr><td><güçlü>mikroskoplar:</güçlü></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>SMLM Mikroskobu</td><td>Cremer Laboratuvarı</td><td></td><td>Dr. S-Y tarafından özel olarak üretildi. Çetin</td></tr></tbody></table>

tek molekül lokalizasyon mikroskobu kullanılarak hücre Çekirdeğinde mutlak dna yoğunluğunun haritalanması

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

Tek Molekül Lokalizasyon Mikroskobu Kullanılarak Hücre Çekirdeğinde Mutlak DNA Yoğunluğunun Haritalanması

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

Biyoloji

Tek molekül mikroskopi, nükleer taşıma yeni bakış açıları sağlanan Yaklaşım.

Tek Molekül Görüntüleme Nükleer Ulaştırma

Biz doğrudan etiketli floresan problar kullanarak 3D DNA FISH ile nükleer mimarisi analiz için sağlam ve çok yönlü protokol açıklar.

Doğrudan Etiketli Problar kullanarak Sağlam 3D DNA BALIK

Tek molekül izleme ile kombine Fotoaktive yerelleştirme mikroskobu (PALM) verir canlı Escherichia coli hücrelerinde protein-DNA etkileşimleri doğrudan gözlem ve ölçümü.

Fotoaktive Tek molekül Takip Kullanma Canlı Bakteriyel Hücrelerde Protein-DNA Etkileşimleri görselleştirme

Floresan işaretli hücrelerin çoğaltılmış görüntü tabanlı analiz sağlayan esnek bir bilişim iş akışı sundu. iş akışı nükleer ve sitoplazmik belirteçleri rakamlarla ve bu bölmeleri arasındaki işaretleyici translokasyonu hesaplar. Prosedürler 96 oyuklu formatta indirek immunofloresan ile işaret tespiti için siRNA ve güvenilir bir yöntem kullanarak hücrelerin pertürbasyon verilmiştir.

Yüksek içerik, Open Source Software tarafından desteklenen Evrensel Mikroskop Verilerden Floresan Göstergeler, Bireysel Hücre Niceleme için iş akışı

Yeni 5-metilsitozin (oksi-mCs), 5-arasında bakterilerde görülen (5hmC), 5-formylcytosine (5 fC) oksitlenmiş formları bulunan ve 5-carboxylcytosine (5caC) farklı DNA değişiklikleri benzersiz işlevsel roller ile temsil edebilir. Yarı nicel bir iş akışı görselleştirme oksi-mCs kayma dağıtım, sinyal şiddeti profil oluşturma ve colocalization için burada kullanılmıştır.

DNA değişiklikler için Immunostaining: Confocal görüntülerin sayısal analiz

3D tek moleküllü lokalizasyon mikroskopisi, yaşayan bakteri hücrelerinde floresan olarak etiketlenmiş proteinlerin mekansal konumlarını ve hareket yörüngelerini araştırmak için kullanılır. Burada açıklanan deneysel ve veri analizi protokolü, sitosolik proteinlerin havuzhalinde ki tek moleküllü yörüngelere dayalı yaygın diffüzif davranışlarını belirler.

Tek Moleküllü İzleme Mikroskobu - Sitosolik Moleküllerin Diffüz Durumlarını Belirlemede Bir Araç

3D çok renkli DNA FISH, 3B korunmuş çekirdekler içinde birden fazla genomik loci görselleştirmek için bir araç temsil eder, kesin olarak tek bir hücre düzeyinde nükleer alan içinde karşılıklı etkileşimleri ve lokalizasyon tanımlayan. Burada, adım protokol bir adım insan birincil hücrelerin geniş bir yelpazede için açıklanmıştır.

3D Çok Renkli DNA FISH Aracı İnsan Birincil Hücrelerde Nükleer Mimari Çalışma

Bu protokol, plazma zarı proteinlerinin nano ölçekli mekânsal organizasyonunu çözmek için tek antikor etiketlemesini (SAL) tanımlar. Tek molekül düzeyinde kümülatif antikor-epitop etkileşimlerini kullanarak, membran SAL (mSAL), yerel epitop dağılımlarını haritalandırırken aynı zamanda doğal hücresel ortamda antikor bağlanma davranışını yakalar.

Tek Antikor Etiketleme Kullanarak Zar Proteinlerinin Tek Moleküllü Lokalizasyon Mikroskopisi

Uzaysal analiz için euchromatin, heterokromatin ve RNAP II'nin tekrarlanabilir haritalanmasını sağlayan üç renkli kromatin tek molekül lokalizasyon mikroskopisi (SMLM) boyama ve analiz protokolü sunuyoruz. Bu protokol, kromatinle ilişkili hedefler dahil olmak üzere yoğun nükleer ortamlarda verimli çok renkli etiketleme sağlar ve güvenilir eşzamanlı tespiti sağlar.

Yoğun Nükleer Ortamda Kromatinin Araştırılması İçin Çok Etiketli Tek Molekül Lokalizasyon Mikroskopi Protokolü

Bu protokol, DNA içsel floresan spektrumlarının örnek hazırlanması, veri toplama ve analizi üzerine ayrıntılı işlemler sağlar.

İçsel Kontrast kullanarak DNA moleküllerinin spektroskopik süper çözünürlüklü görüntülemesi

Bu protokol, izole Arabidopsis çekirdeklerinin tek moleküllü lokalizasyon mikroskopik görüntülemesi için verimli bir iş akışı sunar; optimize edilmiş izolasyon, fiksasyon ve etiketleme kullanılarak kromatin ve RNA Polimeraz II'nin nano ölçekli görselleştirilmesini sağlar. Bu yaklaşım, yüksek çözünürlüklü görüntüleme için diğer kromatin ilişkili proteinlere veya histon modifikasyonlarına kolayca uyarlanabilir.

Arabidopsis çekirdeklerinde tek moleküllü lokalizasyon mikroskopisi için sadeleştirilmiş bir protokol

Keşfedin, tek molekül lokalizasyon mikroskobu ve Voronoi tessellation kullanarak hücre çekirdeğinde mutlak DNA yoğunluğunu haritalama yöntemini. Bu yaklaşım, kromatin mimarisindeki uzamsal kısıtlamaları incelemeyi sağlar.

Tek Molekül Lokalizasyon Mikroskobu Kullanılarak Hücre Çekirdeğinde Mutlak DNA Yoğunluğunun Haritalanması

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