ENZEL - A cryogenic, retrofittable, coincident fluorescence, electron, and ion beam solution for the cryo-electron tomography workflow.

3D Fluorescence Localization in Frozen Cells for Targeted Lamella Milling for Electron Cryo-Tomography

“…Regular wide-field fluorescence microscopy, however, does not bring sufficient resolving power to allow localization and targeting of sparse fluorescence features in the 100-200 nm thick lamella. We are therefore implementing 3D localization using astigmatic imaging in an integrated cryo-fluorescence-FIB-SEM [6,7]. The cryogenic conditions pose two unknown constraints on the localization accuracy: (1) a limited numerical aperture (NA) of the objective lens, and (2) cryogenic immobilization of the fluorescence transition dipole moment of the molecule.…”

“…Our results demonstrate that 3D localization of fluorescent features, from single molecules to fluorescently labeled vesicles, can be achieved at accuracies below the thickness of lamellae required for electron cryo-tomography. Using the aforementioned integrated cryo-fluorescence-FIB-SEM [6], we put our findings to use in the targeted lamella preparation from vitreous cells with fluorescently labeled features. We will discuss further implementation of and prospects for a simplified workflow for the preparation of lamellae with minimized contamination.…”

“…We will discuss further implementation of and prospects for a simplified workflow for the preparation of lamellae with minimized contamination. In the future, the localization methods as presented here, in combination with the integrated cryo-fluorescence-FIB-SEM [6], will allow for correlative super-resolution localization microscopy and electron cryo-tomography [12].…”

“…These methods can however be laborious and prone to contamination of the sample, hindering the acquisition of high-quality electron tomograms. Integration of a light microscope into a cryo-FIB-SEM allows for a simplified workflow to target fluorescent features [4][5][6]. Regular wide-field fluorescence microscopy, however, does not bring sufficient resolving power to allow localization and targeting of sparse fluorescence features in the 100-200 nm thick lamella.…”

Reproducible lamella preparation for electron cryo-tomography by in-situ thickness estimation during fluorescence-guided FIB milling

“…Here, we present a calibration-free method to accurately estimate local lamella thickness in-situ. Our method is based on an integrated cryo-fluorescence-FIB-SEM instrument that allows, unlike commercially available fluorescence microscopes (FM), coincident imaging using FM, FIB or SEM beams [2]. To estimate lamella thickness, we utilise the optical unit in our integrated setup as a highresolution 2D-STEM camera (Fig.…”