The cells were treated with 200 mM calcium chloride directly before plunge freezing. Cells with high GCaMP fluorescence by cryo-FM were targeted for cryo-FIB milling. Electron cryo-tomographic tilt-series were collected on a Titan Krios (FEI) operated at 300 kV using a Quantum energy filter (slit width 20 eV) and a K2 direct electron detector (Gatan) in counting mode at a pixel size of 3.7 angstroms and at a dose rate of ~ 2-4 e-/pixel/second on the detector. Tilt-series were acquired between +/- 60 degrees starting from 0 degrees with 1 degrees increment using SerialEM (Mastronarde, 2005) following a grouped dose-symmetric acquisition with a group size of 4 (Bharat et al., 2018; Hagen et al., 2017), and at -5 micron defocus. A dose of 1.0 e-/square angstroms was applied per image of the tilt-series. The tilt-series frames were aligned and then reordered by tilt angle using IMOD.
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