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Center for Structural Biology

Training

Specimen Preparation Vitrobot Training Module 

Before Training Module 

  1. Watch Grant Jenson Lectures on cryo-specimen preparation.
  2. Read Vitrobot Protocol on CryoEM Core Website. 
  3. Read the policies and rigor reproducibility section of the core website.
  4. Set up an iLAB account, if you don’t have one use this link to setup an account.
  5. The student submits a Vitrobot training request on iLAB or they can contract the Core Director directly.

Training Module 

  1. Overview of Vitrobot and other equipment. 
  2. Overview of TEM grids, plasma cleaner and handling of TEM grids. 
  3. Core Staff shows the students how to operate the Vitrobot while talking through process, student takes notes on handout (Vitrobot Protocol or Quick Start Guide). 
  4. Core Staff show the student how to fill the Vitrobot notebook and transfer the cryogrids into the storage dewar.
  5. Students prepare cryo-grids using old TEM grids and buffer.  Staff watch students and provide feedback or help.
  6. We do not use real samples during the training module.   
  7. Clipping grids is included in this training module.

After Training Module 

  1. Students practice handling grids on their own time. 
  2. Students schedule assessment sessions with Core Staff.  During the assessment, the Core staff watch students operate the Vitrobot and prepare 4 cryogrids using old TEM grids and water.  
  3. Core Staff use Rubrik to score students.  A passing grade of 3 or higher is required.
  4. If a student passes, they can book and operate the Vitrobot without Core Staff.  
  5. If a student fails or is uncomfortable operating the Vitrobot further training sessions with the core staff and reassessment are possible.

 

Talos Arctica Training Module

Before Training Module 

  1. Watch Grant Jenson Lectures on cryoTEM https://cryo-em-course.caltech.edu/unit-1-outline
  2. Read CryoEM 101 Chapter 3 Grid Screening & Evaluation and Chapter 4 Cryo-EM Data Collection (however, all chapters are recommended). https://cryoem101.org/
  3. Visit the cryoEM Core website to learn about the policies, rigor reproducibility, and protocols related to cryoEM. Read the protocols for operating the microscope, SerialEM Lecture, and watch videos on operation of the microscope. 
  4. Set up an iLAB account. https://uncch.ilab.agilent.com/account/login
  5. Setup Globus Account. https://www.globus.org
  6. We recommend that the student watches a person operate the microscope (ideally from their group).

Training Module 

Goals: Learn about the microscope configuration, camera settings and how to use SerialEM to operate the microscope.

Part 1: Core Staff operates the microscope for the student while explaining the process. During this process the student will take notes on the Talos Arctica protocol handout from the core website and is expected to ask questions.

Topics covered during the training session include:

  1. Alignment of the TEM.
  2. Using the Autoloader.
  3. Low-dose imaging.
  4. Assessing sample quality and ice thickness.
  5. Acquiring and saving images.
  6. Collecting montages.
  7. Data collection using the serialEM GUI and scripts.
  8. Optional topics will be covered depending on the goals of the student and what imaging modality they are using, ie. single-particle cryoEM, tomography, microED, cryoTEM.

Part 2: The student will operate the microscope under the supervision of the core staff.

The student will do the following:

  1. Inventory autoloader, load and unload a grid.
  2. Use the flu screen and TIA to quickly look at the grid at low magnification.
  3. Find Eucentric height manually using the stage wobbler.
  4. Take Search, View, and Record images with SerialEM. Save the images as snapshots and mrc format.
  5. Take a low magnification montage of the grid of a using SerialEM
  6. Use the navigator to move the stage to 3 areas of the grid and take images.
  7. Run scripts, map2grids and dose settings.
  8. Setup data collection using BIS method on 3 grid squares.

After Training Module

  1. Student reviews their notes taken during a training session on their own.
  2. Students scheduled an assessment session with the cryoEM Core Staff. Core Staff watch students operate the microscope. Core staff score the students using a check list.
  3. If the student passes they can operate the microscope independently.
  4. If the student has deficiencies or is uncomfortable operating the microscope independently further training sessions with the core staff and reassessment is possible.

Youtube Channel

The UNC-CH CryoEM Core YouTube Channel is committed to providing educational content related to biological cryo-electron microscopy (cryoEM), encompassing specimen preparation and the operation of the electron microscope. We post content on cryoTEM of nanoparticles, SP cryoEM of macromolecular complexes, CryoET, and microED. The content is mainly for students, post-doctoral fellows, and researchers utilizing our core facility. Our goal is to provide information to individuals interested in learning the technical aspects of cryoEM. We recognize that each laboratory, core, or center may have distinct policies, procedures, and equipment. Therefore, viewers are urged to consider these variations when viewing the content on this channel. We strive to provide accurate and reliable information, but we cannot guarantee the completeness of the content.