3D protein localization in frozen cells for targeted lamella milling for electron cryotomography
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cryogenic fluorescence microscopy
focused ion beam scanning electron microscopy
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Integrated cryogenic focused ion beam/scanning electron/fluorescence microscopy (cryo-FIB-SEM-FM) may be crucial to target proteins in frozen cells for lamella milling for electron cryotomography (cryo-ET). The integrated FM allows to select a region of interest for cutting lamella for cryo-ET. Inclusion of a fluorescently labeled protein precisely in this lamella may however require more advanced 3D localization. We have tested fluorescence super-resolution techniques for compatibility with the requirements for integrated cryo-FIB-SEM-FM. Using simulations, we have studied the effect of the cryogenic conditions and limited numerical aperture of the FM objective on the localization and targeting.
In-situ imaging of proteins in cells allows to characterize their native structure, conformation and their interactions with their environment. Cryo-ET enables this, but the proteins of interest need to be within a 100-200 nm thick lamella. Current targeting methods include fiducial markers with laborious transfer between different cryogenic microscopy setups, prone to contaminations on the sample. A simplified, integrated workflow will highly benefit the structural study of proteins in cells.
We use an epi-FM integrated into a cryo-FIB-SEM with a nitrogen micro-cooler, developed by Delmic together with us and an international consortium. In this microscope, the sample can be imaged with FM while the sample is in the position and orientation for FIB milling. Using this setup, we can select regions of interest (ROIs) in the sample to be included in the lamella. To assess targeting of sparse proteins, we have conducted simulations to test 3D localization techniques using point spread function (PSF) shaping techniques compatible with the experimental setup where the numerical aperture of the FM objective is limited by its long working distance.
We demonstrate we can cut lamella in a region selected in the FM images without transfer between different microscope setups, therefore preventing unnecessary contamination. For the 3D localization of sparsely labeled ROIs, we find that the fixed dipole orientation of the fluorophores cannot be ignored, even with the limited NA of the FM objective. We show how the localization precision depends on both the angle of the fluorophore with respect to the optical axis of the FM and the number of fluorophores per ROI. Finally, we discuss the different targeting scenarios relevant for the milling of lamella for cryo-ET.