3234 Microsc. Microanal. 27 (Suppl 1), 2021 doi:10.1017/S1431927621011144 © Microscopy Society of America 2021 Fluorescence-guided lamella fabrication with ENZEL, an integrated cryogenic CLEM solution for the cryo-electron tomography workflow Caspar Jonker 1 , Daan Boltje 2 , Jacob Hoogenboom 3 , Arjen Jakobi 4 , Grant Jensen 5 , Abraham Koster 6 , Mart Last 1 , Jürgen Plitzko 7 , Stefan Raunser 8 , Sebastian Tacke 9 , Roger Wepf 10 and Sander Den Hoedt 1 1 Delmic B.V., Kanaalweg 4, 2326 EB Delft, The Netherlands, United States, 2 Department of Imaging Physics, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands, Delft, Netherlands, 3 Technical University Delft, United States, 4 Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands, United States, 5 Caltech, United States, 6 Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands, United States, 7 Max Planck Institute of Biochemistry, Martinsried, Germany, United States, 8 Max Planck Institute of Molecular Physiology, United States, 9 Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany, United States, 10 Centre for Microscopy and Microanalysis, The University of Queensland, St. Lucia Queensland 4072, Brisbane, Australia, United States Cryogenic electron tomography (Cryo-ET) is an imaging technique used to obtain high resolution 3D reconstructions of biomolecules in their near-native cellular environment. In Cryo-ET, a sample is flash frozen, thinned to the appropriate thickness (100-200 nm) and a tomogram is captured using a cryo transmission electron microscope (TEM). To create the thin section for the acquisition of the tomogram, using a Focused Ion Beam in a Scanning Electron Microscope (FIB/SEM) has become the gold standard. The FIB is used to mill away the surrounding material and create a lamella [1], [2]. Identifying the region of interest (ROI) to mill in the right location is a crucial step, since being off-target for this process could result in milling away your structure of interest. To overcome this, cryo-Fluorescence Microscopy (cryo- FM) is often used to identify the region of interest and avoid ‘blind’ milling. In cryogenic Correlative Light and Electron Microscopy (cryo-CLEM), fluorescent markers are used to label the structures or proteins of interest, which are then found back in the FIB/SEM. Incorporation of a cryo-FM in the cryo-ET workflow brings many challenges. Firstly, transfer of the sample to the cryo-FM before milling (to identify ROIs) and again after milling (to ensure the ROI is not milled away) significantly increases the handling of the sample and thereby increases the risk of contaminating or damaging the sample [3], [4]. Secondly, the correlation of the fluorescence image with the image in the FIB/SEM is not trivial. It requires markers for navigating to the correct spot on the sample and if the overlay of the fluorescence and SEM images is not exactly correct, the ROI could still be missed or milled away [5]. We have developed ENZEL, an inverted widefield fluorescence microscope that can be integrated in dual beam systems that allows simultaneous, coincidence imaging of both FM and SEM. A custom sample stage and micro cooler setup allow the approach of an objective lens from below the sample, directly underneath the electron column polepiece. This setup allows imaging using SEM and FM without separate registration. Fluorescence imaging is especially beneficial to the cryo-ET workflow when targeting specific intracellular compartments or distinctly localized proteins. We illustrate the benefits of this system with several example problem cases. We performed multi-channel fluorescence imaging of plunge-frozen https://doi.org/10.1017/S1431927621011144 Published online by Cambridge University Press