Name: Imaging spettroscopico a super-risoluzione delle molecole di DNA utilizzando contrasto intrinseco (Video) | JoVE Journal
Uploaded: 2026-03-06T13:25:00.000+00:00
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Description: Scopri l'imaging spettroscopico a super-risoluzione del DNA senza marcatori usando la fluorescenza intrinseca. Questo metodo consente l'analisi spettrale di molecole a e doppio filamento preservando le proprietà native.

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</ol>

<table><tbody><tr><td>Filtro di emissione a 488 nm per l'imaging</td><td>Chroma Technology Corp.</td><td>ET525/50M</td><td></td></tr><tr><td>Filtro passa-banda da 532 nm per la calibrazione</td><td>Thorlabs</td><td>FL532-3</td><td></td></tr><tr><td>Filtro a emissione da 532 nm per l'imaging</td><td>Chroma Technology Corp.</td><td>ET620/60M</td><td></td></tr><tr><td>Filtro passa-banda a 580 nm per la calibrazione</td><td>Thorlabs</td><td>FB580-10</td><td></td></tr><tr><td>Filtro passa-banda a 633 nm per la calibrazione</td><td>Thorlabs</td><td>FL632.8-3</td><td></td></tr><tr><td>Filtro passa-banda a 680 nm per la calibrazione</td><td>Thorlabs</td><td>FB680-10</td><td></td></tr><tr><td>Filtro passabanda a 750 nm per la calibrazione</td><td>Thorlabs</td><td>FB750-10</td><td></td></tr><tr><td>Beta-mercaptoetanolo&nbsp;</td><td>Sigma</td><td>M6250</td><td></td></tr><tr><td>Buffer di imaging BME</td><td>NA</td><td>NA</td><td>7 & mu; L GLOX, 7 e mu; L Beta-mercaptoetanolo e 690 &mu; Tampone L glucosio</td></tr><tr><td>Catalasi dal fegato bovino</td><td>Sigma</td><td>C100</td><td></td></tr><tr><td>DABCO (1,4-di-azobiciclo-(2.2.2.)-Ottano)</td><td>Sigma</td><td>D27802</td><td></td></tr><tr><td>Buffer di imaging DABCO</td><td>NA</td><td>NA</td><td>65 mM DABCO+30 mM Solfiti di sodio + 30 mM DTT 1 M&nbsp; nel DNAsi, acqua deionizzata libera da RNAsi. Il solfito di sodio dovrebbe essere disciolto in PBS 10 e volte; a 1 M prima di realizzare il buffer di imaging.</td></tr><tr><td>Deplex del DNA per SMLM spettroscopico</td><td>IDT</td><td>NA</td><td>Coppie a 20 basi doppio filo GC_alter, GC_conti, AT_alter e AT_conti. ad esempio GC_alter ha entrambi i fili da 5' - GCGCGCGCGCGCGCGCGCGCGCGC - 3'; GC_conti ha un filo di 5' - GGGGGGGGGGGGGGGGGGGG - 3' e l'altro come 5' - CCCCCCCCCCCCCCCCCCCCCCCCCC - 3' Tutti i campioni sono nello standard HPLC.</td></tr><tr><td>DNA oligonucleotide/DNA doppio filamento</td><td>IDT</td><td>NA</td><td></td></tr><tr><td>Oligos del DNA per SMLM spettroscopico</td><td>IDT</td><td>NA</td><td>20-base A, G, C, T; catena AC a singolo filamento a 40 basi (A1C1, A5C5, A10C10, A20C20); &nbsp; guanina monofilamento 5, 8, 12, 16 basi. Ad esempio A10C10 è 5'- AAAAAAAAAACCCCC CCCCCAAAAAAAAAA AACCCCCCCCCCCC - 3' Tutti i campioni sono secondo lo standard HPLC.</td></tr><tr><td>Acqua distillata senza DNasi/RNase</td><td>Invitrogen</td><td>10977015</td><td></td></tr><tr><td>DTT (DL-Ditiotreitolo)</td><td>Sigma</td><td>43816</td><td></td></tr><tr><td>GLOX</td><td>NA</td><td>NA</td><td>14 mg di glucosio ossidasi, 50 e mu; L catalasi (17 mg/ml) + 200 & mu; Il tampone L A, che è di 10 mM Tris (pH 8,0) e 50 mM di NaCl.&nbsp;</td></tr><tr><td>Buffer di glucosio</td><td>NA</td><td>NA</td><td>50 mM Tris (pH 8,0), 50 mM di NaCl e 10% di glucosio.</td></tr><tr><td>Glucosio ossidasi</td><td>sigma</td><td>G7141</td><td></td></tr><tr><td>Glucosio, polvere</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>Cloruro di sodio</td><td>Thermo Fisher</td><td>424290010</td><td></td></tr><tr><td>Solfiti di sodio&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>

imaging spettroscopico a super-risoluzione delle molecole di dna utilizzando contrasto intrinseco

Spectroscopic Super-resolution Imaging of DNA Molecules using Intrinsic Contrast

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

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

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Questo video descrive un metodo per visualizzare l'assemblaggio di condensati di proteine di fusione su molecole di DNA in loci specifici utilizzando l'imaging a singola molecola. Questi condensati si formano a causa delle interazioni LCD-LCD e LCD-DBD, che consentono il reclutamento della RNA polimerasi II e una migliore trascrizione genica.

Imaging di una singola molecola per visualizzare l'assemblaggio di condensati di proteine di fusione su tende di DNA

Descriviamo un super-risoluzione metodo di imaging per sondare l'organizzazione strutturale del batterica FtsZ-ring, un apparecchio essenziale per la divisione cellulare. Questo metodo si basa su analisi quantitative di fotoattivati ​​localizzazione microscopia (PALM) immagini e può essere applicata ad altre proteine ​​batteriche citoscheletro.

Super-risoluzione Imaging della Divisione Macchine batterica

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

L'obiettivo generale di questa metodologia è quello di dare le condizioni sperimentali ottimali dalla preparazione del campione per acquisizione di immagini e la ricostruzione al fine di eseguire immagini dSTORM 2D doppio colore dei microtubuli e filamenti intermedi in celle...

I filamenti intermedi e microtubuli con microscopia di ricostruzione ottico stocastico diretto 2-dimensionale di imaging

Un'istruzione dettagliata è descritta su come costruire un altamente propenso spazzato tile (HIST) microscopio e suo utilizzo per l'imaging di singola molecola.

Una guida per costruire un altamente propenso spazzato Tile microscopio per l'esteso campo di vista singola molecola Imaging

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

Il presente protocollo descrive un metodo che misura la densità assoluta del DNA all'interno di nuclei cellulari aderenti utilizzando la tassellatura Voronoi di dati di microscopia di localizzazione di singole molecole, volume noto, dimensione del genoma e stadio del ciclo cellulare.

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

Questo protocollo dimostra l'esecuzione di un saggio a singola molecola per la visualizzazione dal vivo dello svolgimento del DNA mediante elicasi CMG. Descrive (1) la preparazione di un substrato di DNA, (2) la purificazione dell'elicasi CMG di Drosophila melanogaster marcata in fluorescenza, (3) la preparazione di una cella a flusso microfluidico per la microscopia a fluorescenza a riflessione interna totale (TIRF) e (4) il saggio di svolgimento del DNA a singola molecola.

Visualizzazione in tempo reale a singola molecola dello svolgimento del DNA mediante elicasi CMG

Questo protocollo delinea la procedura per eseguire la microscopia di localizzazione a singola molecola profonda e intera cellula (SMLM) utilizzando un microscopio confocale a disco rotante con riassegnazione ottica dei fotoni, consentendo così l'imaging DNA-PAINT senza la necessità di ottica personalizzata o illuminazione complessa.

Imaging a superrisoluzione di cellule intere tramite DNA-PAINT su un disco confocale rotante con riassegnazione ottica dei fotoni

"E-DNA" sensori, reagentless, biosensori elettrochimici che ottengono buoni risultati anche quando sfidato direttamente nel sangue e altre matrici complesse, sono stati adattati per il rilevamento di una vasta gamma di acidi nucleici, proteine ​​e analiti di piccole molecole. Qui vi presentiamo una procedura generale per la fabbricazione e l'uso di sensori di questo tipo.

Fabbricazione di elettrochimica-DNA biosensori per il rilevamento Reagentless di acidi nucleici, proteine ​​e piccole molecole

Scopri l'imaging spettroscopico a super-risoluzione del DNA senza marcatori usando la fluorescenza intrinseca. Questo metodo consente l'analisi spettrale di molecole a e doppio filamento preservando le proprietà native.

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

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