Environment-Tolerant Blinking Fluorophores Enable Dual-Color Super-Resolution Correlative Light and Electron Microscopy
Year: 2026
Type: preprint
Abstract: Super-resolution correlative light and electron microscopy (SR-CLEM) allows mapping biomolecular localization onto native cellular ultrastructure with nanometer precision. However, its broad application remains largely restricted to single-color imaging, mainly because fluorophores undergo fluorescence loss and/or chemical degradation under the harsh conditions required for EM sample preparation. Here, we overcome this barrier with chemically robust fluorophores that exhibit intrinsic, buffer-free blinking across diverse conditions, including osmium tetroxide (OsO 4 )-treated and epoxy-embedded environments, enabling single-molecule localization microscopy (SMLM) directly on EM-prepared sections. Using these probes, we demonstrate the first dual-color SR-CLEM of endogenous proteins on the same ultrathin section. Photophysical analyses attribute OsO 4 -induced fluorescence attenuation primarily to distance-dependent photoinduced electron transfer/FRET quenching and an inner-filter absorption by reduced osmium species, rather than irreversible chemical degradation. This work develops a practical SR-CLEM platform based on a set of tailored organic fluorophores and provides a rational photophysical framework for designing next-generation EM-compatible probes with universal, durable intrinsic blinking properties, enabling a broad range of advanced correlative nanoscopy.
Source: ChemRxiv
Language: English
Subfield: Biophysics
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7212270670