4.8 Article

Carbon Dot Blinking Enables Accurate Molecular Counting at Nanoscale Resolution

Journal

ANALYTICAL CHEMISTRY
Volume 93, Issue 8, Pages 3968-3975

Publisher

AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.0c04885

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Funding

  1. National Natural Science Foundation of China [21874154]
  2. Fundamental Research Funds for the Central Universities [18CX02126A]

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This study developed a new technique for accurate single-molecule counting using spontaneous blinking of carbon dots, achieving high precision with a localization accuracy of 10 nm. The technique was successfully applied to study molecular interactions and distribution in subcellular fractions, as well as G-protein coupled receptors on cell membranes, providing insights into receptor oligomerization, clustering, and ligand-regulated distributions. The adaptation of nanoparticle self-blinking for molecular counting demonstrates the potential of carbon dots as reliable probes for deciphering sub-diffraction structures essential for biological functions.
Accurate counting of single molecules at nanoscale resolution is essential for the study of molecular interactions and distribution in subcellular fractions. By using small-sized carbon dots (CDs), we have now developed a quantitative single-molecule localization microscopy technique (qSMLM) based on spontaneous blinking to count single molecules with a localization precision of 10 nm, which can be accomplished on conventional microscopes without sophisticated laser control. We explore and adapt the blinking of CDs with diverse structures and demonstrate a counting accuracy of >97% at a molecular density of 500 per mu m(2). When applied to G-protein coupled receptors on a cell membrane, we discriminated receptor oligomerization and clustering and revealed ligand-regulated receptor distribution patterns. This is the first example of adapting nanoparticle self-blinking for molecular counting, and this demonstrates the power of CDs as SMLM probes to reliably decipher sub-diffraction structures that mediate crucial biological functions.

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