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Cryo-iCLEM: Cryo correlative light and electron microscopy with immersion objectives
Niko Faul1, Shih-Ya Chen1, Christian Lamberz1
1Technische Universität Darmstadt, Department of Electrical Engineering and Information Technology, Merckstrasse 25, 64283 Darmstadt, Germany.
Journal of Structural Biology
|February 19, 2025
Summary
We developed a novel workflow for correlative cryoimmersion light microscopy and electron cryomicroscopy (cryo-iCLEM). This method preserves sample integrity and ultrastructure, enabling detailed cellular investigation at cryogenic temperatures.
Area of Science:
- Cellular and Molecular Imaging
- Cryo-Electron Microscopy
- Advanced Light Microscopy Techniques
Background:
- Correlative light and electron microscopy (CLEM) is crucial for molecular-level cellular investigation.
- Electron microscopy benefits from cryogenic temperatures, but light microscopy often lacks cryo-compatible immersion objectives.
- Existing cryo-immersion light microscopy (cryo-iLM) techniques have not been integrated into correlative workflows.
Purpose of the Study:
- To present a novel workflow for correlative cryoimmersion light microscopy and electron cryomicroscopy (cryo-iCLEM).
- To demonstrate the compatibility of cryo-iLM with cryo-electron tomography (cryo-ET) and focused ion beam (FIB) milling.
- To showcase the advantages of cryo-iCLEM for high-resolution cellular imaging at cryogenic temperatures.
Main Methods:
- Development of a cryo-iCLEM workflow involving sample embedding in a viscous immersion medium at cryogenic temperatures.
- Examination of samples using a custom cryo-immersion objective for cryo-iLM.
- Dissolution of the immersion medium in liquid ethane post-cryo-iLM, enabling subsequent cryo-electron microscopy (cryo-EM) imaging.
- Application of cryo-electron tomography (cryo-ET) before and after cryo-iLM to assess sample integrity and ultrastructure.
Main Results:
- Cryo-iCLEM maintains ultra-thin, electron-transparent samples mechanically intact, without degrading ultrastructural preservation from plunge-freezing.
- The workflow successfully imaged FIB-milled lamellae, showing that mechanically sensitive samples remain undamaged.
- Embedding in immersion fluid reduces contamination, allowing extended data acquisition (hours) with low fluorophore bleaching rates.
Conclusions:
- Cryo-iCLEM offers a robust method for combining high-resolution light microscopy with cryo-electron microscopy.
- This technique preserves sample integrity and enhances imaging capabilities at cryogenic temperatures.
- The developed workflow is expected to advance various light microscopy techniques within cryo-CLEM applications.
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