Near-Infrared Blinking Carbon Dots Designed for Quantitative Nanoscopy

Blinking carbon dots (CDs) have attracted attention as a probe for single molecule localization microscopy (SMLM), yet quantitative analysis is limited because of inept blinking and low signal-to-noise ratio (SNR). Here we report the design and synthesis of near-infrared (NIR) blinking CDs with a maximum emission of around 750 nm by weaving a nitrogen-doped aromatic backbone with surplus carboxyl groups on the surface. The NIR-CDs allow conjugation to monovalent antibody fragments for labeling and imaging of cellular receptors as well as afford increases of 52% in SNR and 33% in localization precision over visible CDs. Analysis of fluorescent bursts allows for accurate counting of cellular receptors at the nanoscale resolution. Using NIR-CDs-based SMLM, we demonstrate oligomerization and internalization of programmed cell death-ligand 1 by a small molecule inhibitor for checkpoint blockade. Our NIR-CDs can become a generally applicable probe for quantitative nanoscopy in chemistry and biology.