Environment-Tolerant Blinking Fluorophores Enable Dual-Color Super-Resolution Correlative Light and Electron Microscopy
11 affiliations
- 1Max Planck Institute for Polymer Research, Ackermannweg 10, Mainz, Germany, 55128
- 2Frontier Institute of Science and Technology
- 3Xi'an Jiaotong University
- 4Institute of Molecular Biology gGmbH, Ackermannweg 4, Mainz, Germany, 55128
- 5Optical Neuroimaging Unit
- 6Okinawa Institute of Science and Technology Graduate University
- 7Katz Group Centre
- 8University of Alberta
- 9Organic and Carbon Nanomaterials Unit
- 10Madrid Institute for Advanced Studies, IMDEA Nanoscience, C/Faraday 9, Madrid, Spain, 28049
- 11Key Laboratory for Micro-Nano Optoelectronic Devices of Ministry of Education, School of Physics and Electronics, Hunan University, Changsha, 410082, China.
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.
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BibTeX
@misc{zhu2026environmenttolerant,
title = {{Environment-Tolerant Blinking Fluorophores Enable Dual-Color Super-Resolution Correlative Light and Electron Microscopy}},
author = {Xingfu Zhu and Ze-Hua Wu and Xin Zhou and Anke Kaltbeitzel and Yu-Liang Tsai and Márton Gelléri and Christoph Cremer and Bruno M. Humbel and Hilmar Strickfaden and Qiqi Yang and Ingo Lieberwirth and Chen Li and Akimitsu Narita and Klaus Müllen and Johannes Gierschner and Yungui Li and Mischa Bonn and Xiaomin Liu},
howpublished = {ChemRxiv},
year = {2026},
month = sep,
doi = {10.26434/chemrxiv.15002829/v2},
url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15002829/v2},
note = {CC BY},
}
RIS
TY - UNPB
TI - Environment-Tolerant Blinking Fluorophores Enable Dual-Color Super-Resolution Correlative Light and Electron Microscopy
AU - Xingfu Zhu
AU - Ze-Hua Wu
AU - Xin Zhou
AU - Anke Kaltbeitzel
AU - Yu-Liang Tsai
AU - Márton Gelléri
AU - Christoph Cremer
AU - Bruno M. Humbel
AU - Hilmar Strickfaden
AU - Qiqi Yang
AU - Ingo Lieberwirth
AU - Chen Li
AU - Akimitsu Narita
AU - Klaus Müllen
AU - Johannes Gierschner
AU - Yungui Li
AU - Mischa Bonn
AU - Xiaomin Liu
PY - 2026
DA - 2026/09/11
PB - ChemRxiv
DO - 10.26434/chemrxiv.15002829/v2
UR - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15002829/v2
AB - 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.
ER -