tilt series

MRC

121

(3710, 3838)

SIGNED 16 BIT INTEGER

(3.753, 3.753)

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.

Download #files