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Cells ; 8(1)2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-30646582

RESUMO

DNA point accumulation for imaging in nanoscale topography (PAINT) is a rapidly developing fluorescence super-resolution technique, which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20⁻25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using three nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the three targets with 20 nm resolution, and within only 35 minutes acquisition time.


Assuntos
Proteínas Luminescentes/análise , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Anticorpos de Domínio Único/imunologia , Animais , Células COS , Chlorocebus aethiops , Cromatina/química , Cromatina/ultraestrutura , DNA/química , Complexo de Golgi/química , Complexo de Golgi/ultraestrutura , Proteínas Luminescentes/imunologia , Mitocôndrias/química , Mitocôndrias/ultraestrutura
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