A stable cryogenic fluorescence microscope for correlative super-resolution light and electron microscopy

Mojiri, S.; Dobbs, J. M.; Sanchez, R.; Kreshuk, A.; Mahamid, J.; Ries, J.

doi:10.64898/2026.04.18.719389

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bioRxiv · 10.64898/2026.04.18.719389

A stable cryogenic fluorescence microscope for correlative super-resolution light and electron microscopy

Mojiri, S.·Dobbs, J. M.·Sanchez, R.·Kreshuk, A.·Mahamid, J.·Ries, J.

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Abstract

Cryogenic correlative light and electron microscopy (cryo-CLEM) enables visualization of biological specimens with molecular specificity while preserving near-native macromolecular structure. However, the severely limited resolution of conventional cryo-fluorescence microscopes restricts the accuracy of correlation with cryo-electron microscopy. Super-resolution cryogenic CLEM (SR-cryo-CLEM) offers a potential solution, but presents substantial technical challenges, including mechanical instability and ice contamination. Here, we introduce a modular cryogenic light microscope optimized for single-molecule localization microscopy (cryo-SMLM) that mitigates such limitations. The system is constructed primarily from off-the-shelf components, enabling straightforward and cost-effective assembly, and is operated using fully open-source Python software for flexible and customizable control. The mechanically and thermally stabilized architecture, combined with an axial focus-lock system, maintains sample positioning within a standard deviation of 40 nm. Ice contamination is minimized by imaging inside a purged enclosure, enabling prolonged acquisitions. Together, the platform provides robust localization precision, reproducible imaging performance, and an accessible solution for SR-cryo-CLEM.

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Mojiri, S., Dobbs, J. M., Sanchez, R., Kreshuk, A., Mahamid, J., Ries, J.. 2026-04-21. A stable cryogenic fluorescence microscope for correlative super-resolution light and electron microscopy. https://doi.org/10.64898/2026.04.18.719389

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