Long-wavelength excitation of carbon dots with dual-organelle targeting capability for live-cell imaging via STED nanoscopy

Abstract

Mitochondria and lysosomes play important roles in maintaining normal cellular metabolism; thus, visualizing their morphology, structure, and interactions in living cells at the nanometer scale is essential for further understanding their functions. However, this remains challenging because of the current limitations of fluorescent probes, including poor photobleaching resistance, low biocompatibility, and lack of multi-organelle targeting. Herein, we report the development of biocompatible nitrogen-doped carbon dots (CNDs) with long excitation wavelengths, stable fluorescence, and dual-organelle targeting capabilities. The CNDs enter cells predominantly via energy-dependent transport (including clathrin- and caveolae-mediated endocytosis) and free diffusion and subsequently target the interior mitochondria and lysosomes; furthermore, they enable the stimulated emission depletion imaging of these organelles in living cells with resolutions of up to 134 and 55 nm, respectively. This study provides practical guidance for designing dual-organelle targeting fluorescent probes for the dynamic super-resolution imaging of cellular systems.