Photophysical properties and applications of nanographene-based fluorophores in super-resolution imaging

Fluorescence microscopy has advanced significantly for visualizing biological samples, yet traditional techniques are limited by optical diffraction, restricting resolution to above 200 nm and hindering subcellular studies. Super-resolution microscopy (SRM) overcomes these limits, revealing nanoscale cellular dynamics. Recently demonstrated SRM methods, e.g., single-molecule localization microscopy (SMLM) and stimulated emission depletion microscopy (STED) require enhancements to traditional microscopes and depend on specific photophysical properties of fluorescent probes. This thesis investigates the photophysical properties and applications of novel nanographene (NG)-based fluorophores for SRM. Initially, single-molecule fluorescence microscopy and spectroscopy were used to analyze fluorescent contaminants in single-molecule samples, leading to a standardized sample preparation protocol to minimize contamination. Following this protocol, the photophysical properties of NGs at the single-molecule level were characterized, revealing excellent blinking properties for SMLM. Meanwhile, NGs demonstrate tunable optical properties—continuous, bright fluorescence at cluster/ensemble states, and stochastic blinking at ultra-low concentrations/single-molecule levels—making them suitable for both SMLM and STED. Furthermore, NGs exhibited high photostability and unique properties, allowing fluorescence recovery instead of bleaching under near-infrared light (including 775 nm STED beam). This suggests that NGs can enable hours-long, 3D STED imaging, expanding the application of STED microscopy for high-resolution imaging of cellular processes. The fluorescence recovery properties were characterized and possible mechanism was discussed. Inspired by the fluorescence recovery mechanism, a blinking mechanism of NGs at the single-molecule level was proposed. Understanding the blinking mechanism provides a foundation for designing new fluorophores tailored to specific applications.