Cryo-EM Training - Basic (level I & II)

(Each session is set 2 - 2.5 hours)


Chen Xu






The goal of the training we offer is to help a new user coming to EM field to learn some basic knowledge and skill of Cryo-EM. Hopefully, at the end of these two levels of training sessions, you will be able to screen your negative stained specimen and cryo grids on a TEM scope without too much difficult.


You are not required to have experience with TEM operation. Certainly, we could progress faster if you have some basic knowledge about a TEM. The only requirement to you, the fresh trainee, is to have a spirit of commitment. You need to be determined to learn this new technique. If you think to have your data collected while in your training session and have your project done half way, you should not attend the training session that we offer. You will be wasting your own time, let alone ours. Usually, if an user doesn’t take good note during training session and doesn’t come back to practise on microscope, that is a good hint that the user is not serious at it. Operating a transmission electron microscope is very much like driving a car - you need to be serious and to practise if you really want to learn it!

This document lists the training contents that these two-level, 8 sessions will cover.

Level I - Basic TEM Operation (4 sessions)

Session I - 1

  • Prepare continous carbon film grid

  • Introduction to TEM; brief history; compare to X-ray as beam source

  • Basic TEM Operation on Tecnai G2 system

  • User Interface & Vacuum system

Session I - 2

  • Specimen rod insertion and retraction

  • Tuning microscope (Eucentricity, gun alignment, filament saturation, beam-tilt pivot points, rotation center)

  • C2 aperture centering, C2 lens stigmation; Obj. aperture centering, Obj lens stigmation

  • Correct side of beam spreading

  • Demo and instroduce CTF concept

Session I - 3

  • Negative Stain sample preparation

  • Refreshing microscope tuning procedure

  • Introduce digital cameras - hardware component, background preparation, pixelsize at levels of detector and specimen

  • Introduce DQE, and latest status of direct electron detectors

  • Negative stain specimen imaging using CCD

Session I - 4

  • Supervising practice of users for negative stain and scope operation

  • Assist users’ operation and answer questions

  • Demo and explain cryo-cycle procedure to condition the column

Level II - Cryo TEM Operation (4 sessions)

Session II - 1

  • Prepare side-entry cryo holder, explain dry pumping station - warm-up & zeolite cycle

  • Demo and explain FEI Virobot operation

  • Explain holey grid pre-treament - bake, wash & electron beam irradiation

  • Demo full procedure for Cryo freezing EM grids

  • explain the Ethane tank operation

Session II - 2

  • Low Dose TEM operation, explain low dose concept and setup

  • Indroduce and explain shutter control on TEM system

  • CCD imaging with Tecnai Low Dose software

Session II - 3

  • Cryo grid loading to cryo holder on transfer station

  • Cryo holder insertion & retraction, explain airlock pumping mechanism

  • Demo and explain vacuum condition and how to quiet LN2 bubbling

  • Go through whole process from sample loading to Low Dose imaging

  • Explain ice condition

Session II - 4

  • Supervising Cryo Session for user practice

  • Debugging ice condition, cryo-cycle

  • Supervising warm-up and baking cryo holder