Name: İçsel Kontrast kullanarak DNA moleküllerinin spektroskopik süper çözünürlüklü görüntülemesi (Video) | JoVE Journal
Uploaded: 2026-03-06T13:25:00.000+00:00
Duration: 9 min 19 s
Description: Öğrenin, DNA moleküllerinin içsel floresansını kullanarak etiketsiz süper çözünürlüklü spektroskopik görüntüleme protokolünü. Bu yöntem, yerel optik özellikleri koruyarak yüksek çözünürlüklü analiz sağlar.

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<li>Davis, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RainbowSTORM%3A+an+open-source+ImageJ+plug-in+for+spectroscopic+single-molecule+localization+microscopy+%28sSMLM%29+data+analysis+and+image+reconstruction%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">RainbowSTORM: an open-source ImageJ plug-in for spectroscopic single-molecule localization microscopy (sSMLM) data analysis and image reconstruction.</a> Bioinformatics. 36 (19), 4972-4974 (2020).</li>
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<li>Varejão, N., Reverter, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+intrinsic+fluorescence+to+measure+protein+stability+upon+thermal+and+chemical+denaturation%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Using intrinsic fluorescence to measure protein stability upon thermal and chemical denaturation.</a> Methods Mol Biol. 2581, 229-241 (2023).</li>
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</ol>

<table><tbody><tr><td>görüntüleme için 488 nm emisyon filtresi</td><td>Chroma Technology Corp.</td><td>ET525/50M</td><td></td></tr><tr><td>Kalibrasyon için 532 nm bant geçirici filtre</td><td>Thorlabs</td><td>FL532-3</td><td></td></tr><tr><td>görüntüleme için 532 nm emisyon filtresi</td><td>Chroma Technology Corp.</td><td>ET620/60M</td><td></td></tr><tr><td>Kalibrasyon için 580 nm bant geçişli filtre</td><td>Thorlabs</td><td>FB580-10</td><td></td></tr><tr><td>Kalibrasyon için 633 nm bant geçişli filtre</td><td>Thorlabs</td><td>FL632.8-3</td><td></td></tr><tr><td>Kalibrasyon için 680 nm bant geçişli filtre</td><td>Thorlabs</td><td>FB680-10</td><td></td></tr><tr><td>Kalibrasyon için 750 nm bant geçişli filtre</td><td>Thorlabs</td><td>FB750-10</td><td></td></tr><tr><td>Beta-merkaptoetanol&nbsp;</td><td>Sigma</td><td>M6250</td><td></td></tr><tr><td>BME görüntüleme tamponu</td><td>NA</td><td>NA</td><td>7 &mu; L GLOX, 7 &mu; L Beta-merkaptoetanol ve 690 &mu; L glikoz tamponu</td></tr><tr><td>Sığır karaciğerinden katalaz</td><td>Sigma</td><td>C100</td><td></td></tr><tr><td>DABCO (1,4-di-azobisicyclo-(2.2.2.)-oktan)</td><td>Sigma</td><td>D27802</td><td></td></tr><tr><td>DABCO görüntüleme tamponu</td><td>NA</td><td>NA</td><td>65 mM DABCO+30 mM Sodyum sülfit + 30 mM DTT 1 M&nbsp; DNAse'de, RNAse serbest deionize su. Sodyum sülfit PBS ile 10 kez çözünmelidir; görüntüleme tamponunu oluşturmadan önce 1 M'ye kadar devam eder.</td></tr><tr><td>Spektroskopik SMLM için DNA deplexi</td><td>IDT</td><td>NA</td><td>20 tabanlı çift çift zincirli GC_alter, GC_conti, AT_alter ve AT_conti. Örneğin GC_alter her iki iplik de 5' olarak - GCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCCGCGCGCCGCGCGCGCGCGCGCG GC_conti bir ipliği 5' uzunluğunda - GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG diğeri ise 5' olarak - CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC - 3' Tüm örnekler HPLC standardındadır.</td></tr><tr><td>DNA oligonukleotid/çift zincirli DNA</td><td>IDT</td><td>NA</td><td></td></tr><tr><td>Spektroskopik SMLM için DNA oligoları</td><td>IDT</td><td>NA</td><td>20 tabanlı A, G, C, T; 40 tabanlı tek zincirli AC zinciri (A1C1, A5C5, A10C10, A20C20); &nbsp; tek zincirli 5-, 8-, 12-, 16-bazlı Guanine. Örneğin A10C10 is 5'- AAAAAAAACCCCC CCCCCAAAAAAAA AACCCCCCCCCC - 3' Tüm örnekler HPLC standardındadır.</td></tr><tr><td>DNaz/RNase-Serbest Damıtılmış Su</td><td>Invitrogen</td><td>10977015</td><td></td></tr><tr><td>DTT (DL-Ditiothreitol)</td><td>Sigma</td><td>43816</td><td></td></tr><tr><td>GLOX</td><td>NA</td><td>NA</td><td>14 mg glikoz oksidaz, 50 &mu; L katalaz (17 mg/ml) + 200 &mu; L tampon A, yani 10 mM Tris (pH 8.0) ve 50 mM NaCl.&nbsp;</td></tr><tr><td>Glikoz tamponu</td><td>NA</td><td>NA</td><td>50 mM Tris (pH 8.0), 50 mM NaCl ve %10 glikoz.&nbsp;</td></tr><tr><td>Glukoz oksidaz</td><td>Sigma</td><td>G7141</td><td></td></tr><tr><td>Glikoz, toz</td><td>Thermo Fisher</td><td>15023021</td><td></td></tr><tr><td>PBS (10X)</td><td>Thermo Fisher</td><td>J62036. K3</td><td></td></tr><tr><td>PBS (1X)</td><td>Gibco</td><td>10010023</td><td></td></tr><tr><td>Sodyum klorür</td><td>Thermo Fisher</td><td>424290010</td><td></td></tr><tr><td>Sodyum sülfit&nbsp;</td><td>Sigma</td><td>S0505</td><td></td></tr><tr><td>Tris (1M) pH 8.0</td><td>Invitrogen</td><td>AM9856</td><td></td></tr></tbody></table>

İçsel kontrast kullanarak dna moleküllerinin spektroskopik süper çözünürlüklü görüntülemesi

Spectroscopic Super-resolution Imaging of DNA Molecules using Intrinsic Contrast

İçsel Kontrast kullanarak DNA moleküllerinin spektroskopik süper çözünürlüklü görüntülemesi

Super-resolution imaging has revolutionized biological research by revealing structural details at the nanoscale. Most optical super-resolution methods ...

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JoVE Journal

Bioengineering

Bu videoda, tek moleküllü görüntüleme kullanarak belirli lokuslarda DNA molekülleri üzerinde füzyon proteini yoğunlaşmalarının montajını görselleştirmek için bir yöntem açıklanmaktadır. Bu kondensatlar, RNA polimeraz II'nin işe alınmasına ve gelişmiş gen transkripsiyonuna izin veren LCD-LCD ve LCD-DBD etkileşimleri nedeniyle oluşur.

DNA perdeleri üzerinde füzyon proteini kondensatlarının montajını görselleştirmek için tek moleküllü görüntüleme

Biz bakteriyel FtsZ-ring yapısal organizasyonu, hücre bölünmesi için gerekli bir aparat soruşturma için görüntüleme yöntemi bir süper-çözünürlük tarif. Bu yöntem, fotoaktive yerelleştirme mikroskobu (PALM) kantitatif görüntü analizi dayanır ve diğer bakteri sitoskeletal proteinleri ile uygulanabilir.

Bakteriyel Bölümü Makine Super çözünürlüklü Görüntüleme

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

Bu metodoloji genel amacı en iyi deneysel koşullar numune hazırlama resim alma ve yeniden yapılanma için 2D çift renk dSTORM görüntülerini mikrotübüller ve ara filamentler sabit hücrelerde gerçekleştirmek için...

Ara filamentler ve mikrotübüller 2 boyutlu doğrudan Stokastik optik imar mikroskobu ile görüntüleme

Ayrıntılı bir talimat nasıl son derece eğimli kurmak için kiremit (HIST) mikroskop ve onun kullanım için tek molekül düşsel süpürüldü açıklanmıştır.

Son derece eğimli oluşturmak için bir kılavuz döşeme mikroskop genişletilmiş görünümü alan tek molekül görüntüleme için süpürüldü.

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

Mevcut protokol, tek moleküllü lokalizasyon mikroskobu verilerinin, bilinen hacmin, genom boyutunun ve hücre döngüsü aşamasının Voronoi mozaiklemesini kullanarak yapışık hücre çekirdekleri içindeki mutlak DNA yoğunluklarını ölçen bir yöntemi açıklamaktadır.

Tek Molekül Lokalizasyon Mikroskobu Kullanılarak Hücre Çekirdeğinde Mutlak DNA Yoğunluğunun Haritalanması

Bu protokol, CMG sarmal ile çözülen DNA'nın canlı görselleştirilmesi için tek moleküllü bir testin gerçekleştirilmesini gösterir. (1) bir DNA substratının hazırlanmasını, (2) floresan etiketli Drosophila melanogaster CMG helikazının saflaştırılmasını, (3) toplam iç yansıma floresan (TIRF) mikroskobu için bir mikroakışkan akış hücresinin hazırlanmasını ve (4) tek moleküllü DNA çözme tahlilini açıklar.

CMG Helikaz ile DNA Çözmenin Tek Moleküllü Gerçek Zamanlı Görselleştirilmesi

Bu protokol, optik foton yeniden atamalı olan dönen disk konfokal mikroskobu kullanarak derin, tam hücre tek moleküllü lokalizasyon mikroskopisi (SMLM) uygulanma prosedürünü özetler; böylece özel optik veya karmaşık aydınlatmaya ihtiyaç olmadan DNA-PAINT görüntüleme mümkün olur.

Optik foton yeniden atamalı dönen disk konfokal üzerinde DNA-PAINT ile tüm hücre süper çözünürlüklü görüntüleme

"E-DNA" sensörleri, reagentless, kan ve diğer karmaşık matrisler doğrudan meydan bile iyi bir performans elektrokimyasal biyosensörler, nükleik asit, protein ve küçük molekül analitler geniş bir algılama için adapte edilmiştir. Burada bu tür sensörlerin üretimi ve kullanımı için genel bir prosedür mevcut.

Nükleik Asitler, Proteinler ve Küçük Moleküller Reagentless Algılama Elektrokimyasal DNA Biyosensörler Fabrikasyon

Öğrenin, DNA moleküllerinin içsel floresansını kullanarak etiketsiz süper çözünürlüklü spektroskopik görüntüleme protokolünü. Bu yöntem, yerel optik özellikleri koruyarak yüksek çözünürlüklü analiz sağlar.

İçsel Kontrast kullanarak DNA moleküllerinin spektroskopik süper çözünürlüklü görüntülemesi

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