The DNA-PAINT palette: a comprehensive performance analysis of fluorescent dyes.
Nat Methods
; 21(9): 1755-1762, 2024 Sep.
Article
em En
| MEDLINE
| ID: mdl-39112798
ABSTRACT
DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a super-resolution fluorescence microscopy technique that achieves single-molecule 'blinking' by transient DNA hybridization. Despite blinking kinetics being largely independent of fluorescent dye choice, the dye employed substantially affects measurement quality. Thus far, there has been no systematic overview of dye performance for DNA-PAINT. Here we defined four key parameters characterizing performance brightness, signal-to-background ratio, DNA-PAINT docking site damage and off-target signal. We then analyzed 18 fluorescent dyes in three spectral regions and examined them both in DNA origami nanostructures, establishing a reference standard, and in a cellular environment, targeting the nuclear pore complex protein Nup96. Finally, having identified several well-performing dyes for each excitation wavelength, we conducted simultaneous three-color DNA-PAINT combined with Exchange-PAINT to image six protein targets in neurons at ~16 nm resolution in less than 2 h. We thus provide guidelines for DNA-PAINT dye selection and evaluation and an overview of performances of commonly used dyes.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA
/
Corantes Fluorescentes
/
Microscopia de Fluorescência
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Nat Methods
Ano de publicação:
2024
Tipo de documento:
Article