Super-Resolved Integrated Cryo-Fluorescence Imaging for Accurate and Precise Cryo-ET Targeting on Lamellae

Cryo focused ion beam (cryo-FIB) milling is a powerful sample preparation technique, expanding the range of biological samples accessible to cryo-electron tomography (cryo-ET). METEOR 2.0 is Delmic’s next-generation integrated cryo-fluorescence microscope (cryo-FM), leveraging higher sensitivity and faster speeds to enable cryo-fluorescence imaging in tandem with cryo-FIB milling - enabling fluorescence-guided lamella preparation and fluorescence re-mapping throughout the milling process [1,2]. METEOR 2.0 incorporates high numerical aperture air objectives to maximize effective resolution and LED-based imaging to avoid the devitrifying potential of laser-based imaging. To further enhance the effective resolution of integrated cryo-FM imaging, we investigated its combination with a super-resolving technique, enhanced Super-Resolution Radial Fluctuations (eSRRF). Super-resolution has been demonstrated on stand-alone cryo-FMs [3,4], however it has not been demonstrated in an integrated cryo-FM system to date. eSRRF is a post-processing technique applied to fluorescence timelapses to extract super-resolved information [5,6]. Using METEOR 2.0, we acquired cryogenic fluorescence timelapses of DNA-origami nanorulers with precisely defined fluorophore separations. Applying eSRRF post-processing yielded a 2-fold improvement in lateral resolution compared to unprocessed widefield imaging. This demonstrates how eSRRF can be applied to integrated-FM-acquired data to overcome the diffraction barrier and enhance the system’s effective resolution. As a proof-of-concept, we applied the METEOR 2.0 - eSRRF workflow to cellular lamellae. METEOR 2.0’s fast dual-channel sample mapping allowed efficient selection of HeLa cells bearing targetable mRFP-GFP-LC3 puncta for cryo-FIB milling. Its high sensitivity imaging revealed colocalizing fluorescent puncta clearly in polished lamellae. eSRRF processing of fluorescence timelapses of these lamellae sharpened structures and improved peak separation, allowing more confident identification of colocalizing puncta. Super-resolved fluorescence signals correlated with organelle ultrastructure observed via cryo-transmission electron microscopy (cryo-TEM), closely aligning with their size and shape. This demonstrates how eSRRF can be applied to cellular lamellae to make the fluorescence data more closely align with what we observe in TEM. This work demonstrates the power of integrated cryo-FM setups and their combination with super-resolving techniques, and is the first demonstration of super-resolution in an integrated cryo-FM setup. This integrated cryo-FM and eSRRF workflow enables high-confidence cryo-ET targeting through accurate and precise post-milling fluorescence mapping on lamellae. Furthermore, the enhanced peak separation achieved with this workflow strengthens target validation, by resolving whether colocalized signals represent true colocalization or sub-diffraction limit proximity.