Carbon Dot Blinking Enables Accurate Molecular Counting at Nanoscale Resolution

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.

Automated summary

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.