A cryo-preparation approach for immuno-fluorescence labelling of cell cultures and CLEM

Session

LSA.3 - Applications for imaging sub-cellular events at high resolution

Authors

Nicolas Schilling (1), Dr. Andres Kaech (1), Johannes Riemann (1), Dr. Jana Doehner (1), Dr. Jose Maria Mateos Melero (1), Dr. Urs Ziegler (1)

Affiliations

1. Center for Microscopy and Image Analysis

Keywords

CLEM, Cryo-prepraration, Super-resolution microscopy, high-pressure freezing, transmission electron microscopy, Rehydration

Abstract text

Routine fixation protocols for immuno-fluorescence labelling are insufficient to preserve the ultra-structure at the resolution accessible with confocal laser scanning and super resolution light microscopy. Cryo-preparation including high-pressure freezing and freeze-substitution offers superior structural preservation, but immuno-fluorescence labelling remains incompatible. However, combining this preparation approach with rehydration enables subsequent immuno-fluorescence labelling while preserving the ultra-structure in its near native state. We established optimized protocols for freeze-substitution and rehydration in order to provide a routine-based method leading to enhanced structural preservation and still being compatible with immuno-fluorescence labelling. Freeze-substitution with 0.1% glutaraldehyde and 0.01% OsO₄ in water-free acetone was combined with initial rehydration at -60°C with 6.5% H₂O. Final and full rehydration was performed at 0°C. Using this approach, extraction of cells was reduced by ~50% compared to standard room temperature fixation and processing. However, the labelling efficiency was reduced due the enhanced structural preservation. Incubation times of antibodies needed to be extended for satisfying fluorescence signals. Imaged cells were finally high-pressure frozen again, freeze-substituted and embedded using standard protocols and the ultrastructure assessed by transmission electron microscopy was correlated to light microscopy data.