West/Midwest Consortium

The goal of the West/Midwest Consortium for High-Resolution Cryo Electron Microscopy (cryoEM) is to offer our consortium users free access to an existing core facility at UCLA

Instructions: Imaging and Data Handling

While our staff prepares the grids for loading into the instrument, other staff members will set up SerialEM for data collection. Our staff will utilize their own accounts to access the Krios computer while serving users. 

First, the atlas of the grid is taken. Then, squares with thin ice are chosen for magnified images in order to pick ideal imaging regions. Finally, targets with thin ice and good particle distribution are selected by the user for final imaging. 

On-site staff will set up TeamViewer/VNC software for consortium users to access and control the Titan Krios TEM computer remotely if they are not collecting data in-person.

About Imaging Conditions Details:

The summit K2 camera is operated with the automation software SerialEM, working continuously 24/7 at rate of 1 – 3 images per minute, producing around 1000 – 3000 images per day. Data is recorded at a normal magnification of 130 kx with a pixel size of ~1.07 Å/pixel on the specimen. Image stacks are recorded at 5 frames/sec for ~6 seconds, with the optimal dose rate and total dosage being set to ~8 e/pix/sec and ~40 e respectively. After drift correction with Motioncor2, two averaged micrographs with and without dose-weighting will be saved for each image stack. 

In order to minimize beam-tilt artifacts, EICN staff uses coma-free alignment to align the microscope and to minimize the beam tilt by aligning the beam as precisely as possible. The beam is deliberately tilted left and right along the X-axis, the defocus in the Fourier transformations of the two images is measured, and the X-tilt that equalizes left and righted focus is computed. This calibration is also done along the Y-axis. 

A parallel beam is used to avoid the type of beam tilt introduced by divergence of the beam and to insure constant magnification for images at different defocus levels. The performance of the microscope is regularly monitored by checking Thon rings in the power spectrum of carbon images. To recover the bad pixels generated by electrons, the camera sensor of the K2 DED is conditioned by raising the temperature to 50°C overnight every week.

All data collected will be saved onto two external hard drives, one as backup, and be sent back to the respective PI/key personnel by EICN staff. Upon confirmation of receipt of the data by the user, EICN will delete their backup copy. We encourage the receiving party to make another copy of the data for their records. The options available to users for data transfer are described below.

Data Transfer Options:

After the session, all data will be stored on external disks. Users have several options available to them for data transfer:

  1. Users may bring their own storage device for storing their data on
  2. Users may store their data on one of EICN’s storage disks. The WMC User Coordinator will then arrange for the disk to be mailed to the user’s lab.
  3. Online transfer