Name: Spectroscopische superresolutiebeeldvorming van DNA-moleculen met intrinsiek contrast (Video) | JoVE Journal
Uploaded: 2026-03-06T13:25:00.000+00:00
Duration: 9 min 19 s
Description: Leer spectroscopische superresolutiebeeldvorming van DNA-moleculen met intrinsiek contrast. Deze labelvrije methode met sSMLM behoudt native moleculaire eigenschappen en voorkomt artefacten van fluorescentielabels.

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</ol>

<table><tbody><tr><td>488 nm emissiefilter voor beeldvorming</td><td>Chroma Technology Corp.</td><td>ET525/50M</td><td></td></tr><tr><td>532 nm bandpass filter voor kalibratie</td><td>Thorlabs</td><td>FL532-3</td><td></td></tr><tr><td>532 nm emissiefilter voor beeldvorming</td><td>Chroma Technology Corp.</td><td>ET620/60M</td><td></td></tr><tr><td>580 nm bandpass filter voor kalibratie</td><td>Thorlabs</td><td>FB580-10</td><td></td></tr><tr><td>633 nm bandpass filter voor kalibratie</td><td>Thorlabs</td><td>FL632.8-3</td><td></td></tr><tr><td>680 nm bandpass filter voor kalibratie</td><td>Thorlabs</td><td>FB680-10</td><td></td></tr><tr><td>750 nm bandpass filter voor kalibratie</td><td>Thorlabs</td><td>FB750-10</td><td></td></tr><tr><td>Beta-mercaptoethanol&nbsp;</td><td>Sigma</td><td>M6250</td><td></td></tr><tr><td>BME beeldvormingsbuffer</td><td>NA</td><td>NA</td><td>7 &mu;L GLOX, 7 &mu;L Beta-mercaptoethanol en 690 &mu;L glucose buffer</td></tr><tr><td>Catalase uit runderlever</td><td>Sigma</td><td>C100</td><td></td></tr><tr><td>DABCO (1,4-di-azobicyclo-(2.2.2.)-octaan)</td><td>Sigma</td><td>D27802</td><td></td></tr><tr><td>DABCO beeldvormingsbuffer</td><td>NA</td><td>NA</td><td>65 mM DABCO+30 mM Natriumsulfiet + 30 mM DTT 1 M&nbsp; in DNAse, RNAse vrij gedeioniseerd water. Het natriumsulfiet moet worden opgelost in PBS 10&times; tot 1 M voordat de beeldvormingsbuffer wordt gemaakt.</td></tr><tr><td>DNA deplex voor spectroscopische SMLM</td><td>IDT</td><td>NA</td><td>20-basenpaar dubbelstrengs GC_alter, GC_conti, AT_alter en AT_conti. Bijv. GC_alter heeft beide strengen als 5&rsquo; - GCGCGCGCGCGCGCGCGCGC - 3&rsquo;; GC_conti heeft de ene streng als 5&rsquo; - GGGGGGGGGGGGGGGGGGGG - 3&rsquo; en de andere als 5&rsquo; - CCCCCCCCCCCCCCCCCCCCCC - 3&rsquo; Alle monsters zijn in HPLC-standaard.</td></tr><tr><td>DNA oligonucleotide/dubbelstrengs DNA</td><td>IDT</td><td>NA</td><td></td></tr><tr><td>DNA oligo's voor spectroscopische SMLM</td><td>IDT</td><td>NA</td><td>20-base A, G, C, T; 40-base enkelstrengs AC-keten (A1C1, A5C5, A10C10, A20C20);&nbsp; enkele streng 5-, 8-, 12-, 16- base Guanine. Bijv. A10C10 is 5'- AAAAAAAAAACCCCC CCCCCAAAAAAAA AACCCCCCCCCC - 3' Alle monsters zijn in HPLC-standaard.</td></tr><tr><td>DNase/RNase-vrij gedestilleerd water</td><td>Invitrogen</td><td>10977015</td><td></td></tr><tr><td>DTT (DL-Dithiothreitol)</td><td>Sigma</td><td>43816</td><td></td></tr><tr><td>GLOX</td><td>NA</td><td>NA</td><td>14 mg glucoseoxidase, 50 &mu;L catalase (17 mg/ml) + 200 &mu;L buffer A, dat is 10 mM Tris (pH 8.0) en 50 mM NaCl.&nbsp;</td></tr><tr><td>glucose buffer</td><td>NA</td><td>NA</td><td>50 mM Tris (pH 8.0), 50 mM NaCl en 10% glucose.&nbsp;</td></tr><tr><td>glucose oxidase</td><td>sigma</td><td>G7141</td><td></td></tr><tr><td>Glucose, poeder</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>Natriumchloride</td><td>Thermo Fisher</td><td>424290010</td><td></td></tr><tr><td>Natriumsulfiet&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>

spectroscopische superresolutiebeeldvorming van dna-moleculen met intrinsiek contrast

Spectroscopic Super-resolution Imaging of DNA Molecules using Intrinsic Contrast

Spectroscopische superresolutiebeeldvorming van DNA-moleculen met intrinsiek contrast

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

spectroscopic-super-resolution-imaging-dna-molecules-using-intrinsic

Research

JoVE Journal

Bioengineering

Bio-engineering

This video describes a method to visualize the assembly of fusion protein condensates on DNA molecules at specific loci using single-molecule imaging. These condensates are formed due to LCD-LCD and LCD-DBD interactions, which allow for the recruitment of RNA polymerase II and enhanced gene transcription.

Single-Molecule Imaging to Visualize Fusion Protein Condensates Assembly on DNA Curtains

We beschrijven een super-resolutie beeldvorming methode om de structurele organisatie van de bacteriële FtsZ-ring, een essentieel inrichting voor celdeling sonde. Deze methode is gebaseerd op kwantitatieve analyses van fotogeactiveerde lokalisatie microscopie (PALM) beelden en kan worden toegepast op andere bacteriële cytoskeleteiwitten.

Super-resolution Imaging van de bacteriële Division Machinery

Onlangs ontdekte geoxideerde vormen van 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) en 5-carboxylcytosine (5caC) kan verschillende DNA wijzigingen met unieke functionele rollen. Hier wordt een semi-kwantitatieve workflow voor visualisatie van oxi-mCs ruimtelijke verdeling, signaal intensiteit profilering en colocalization omschreven.

Immunokleuring voor DNA wijzigingen: computationele analyse van Confocal beelden

Het algemene doel van deze methodiek is geven de optimale proefomstandigheden van monstervoorbereiding Beeldacquisitie en wederopbouw om uit te voeren van 2D dubbele dSTORM kleurenafbeeldingen van microtubuli en intermediaire filamenten in vaste...

Imaging Intermediaire filamenten en microtubuli met 2-dimensionale directe stochastische optische wederopbouw microscopie

Een gedetailleerde instructies wordt beschreven op het bouwen van een sterk geneigd geveegd Tegel (HIST.) Microscoop en het gebruik ervan voor single-molecuul imaging.

Een gids voor het bouwen van een sterk geneigd geveegd tegel Microscoop voor uitgebreide veld-of-view Single-molecuul Imaging

3D multicolor DNA FISH vertegenwoordigt een hulpmiddel om meerdere genomische loci binnen 3D bewaarde kernen te visualiseren, ondubbelzinnig definiëren d.m.v. hun wederzijdse interacties en lokalisatie binnen de nucleaire ruimte op één celniveau. Hier wordt een stap voor stap protocol beschreven voor een breed spectrum van menselijke primaire cellen.

3D Multicolor DNA FISH Tool om nucleaire architectuur in menselijke primaire cellen te bestuderen

Het huidige protocol beschrijft een methode voor het meten van absolute DNA-dichtheden in adherente celkernen met behulp van Voronoi-tessellatie van lokalisatiemicroscopiegegevens van één molecuul, bekend volume, genoomgrootte en celcyclusstadium.

Absolute DNA-dichtheid in celkernen in kaart brengen met behulp van lokalisatiemicroscopie met één molecuul

Dit protocol demonstreert het uitvoeren van een single-molecule assay voor live visualisatie van DNA-afwikkeling door CMG helicase. Het beschrijft (1) het prepareren van een DNA-substraat, (2) het zuiveren van fluorescerend gelabelde Drosophila melanogaster CMG-helicase, (3) het voorbereiden van een microfluïdische flowcel voor totale interne reflectiefluorescentie (TIRF) microscopie, en (4) de single-molecule DNA-afwikkeltest.

Real-time visualisatie van DNA met één molecuul door CMG Helicase

Dit protocol beschrijft de procedure voor het uitvoeren van diepe, whole-cell single-molecule localization microscopy (SMLM) met behulp van een draaiende schijf confocale microscoop met optische fotonherplaatsing, waardoor DNA-PAINT-beeldvorming mogelijk is zonder de noodzaak van aangepaste optica of complexe verlichting.

Superresolutiebeeldvorming van hele cellen via DNA-PAINT op een draaiende schijf confocaal met optische fotonhertoewijzing

"E-DNA" sensoren, reagentless hebben elektrochemische biosensoren die goed presteren, zelfs wanneer direct uitgedaagd in het bloed en andere complexe matrices, aangepast aan de detectie van een breed scala van nucleïnezuur, eiwitten en kleine moleculen analyten. Hier presenteren we een algemene procedure voor de fabricage en het gebruik van dergelijke sensoren.

Fabricatie van elektrochemische-DNA Biosensoren voor de Reagentless detectie van nucleïnezuren, eiwitten en kleine moleculen

Leer spectroscopische superresolutiebeeldvorming van DNA-moleculen met intrinsiek contrast. Deze labelvrije methode met sSMLM behoudt native moleculaire eigenschappen en voorkomt artefacten van fluorescentielabels.

Spectroscopische superresolutiebeeldvorming van DNA-moleculen met intrinsiek contrast

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