Cryo-Electron Microscopy Laboratory Workflow & Supplies
Expert staff at NYU Langone’s Cryo–Electron Microscopy Laboratory use modern cryo–electron microscopes to screen and obtain high-resolution images of your samples.
The typical workflow is as follows:
- Obtain or prepare a sample to image, for instance, a column-purified solution of a macromolecular complex.
- Check this sample by negative-stain electron microscopy using, for example, Thermo Scientific Talos™ L120C microscope in the Microscopy Laboratory.
- Freeze electron microscopy grids containing your sample using, for example, our Thermo Scientific Vitrobot™ Mark IV or Leica EM GP. We can advise or help you operate the microscopes.
- Store your grids in our liquid nitrogen dewars if desired.
- Bring us your grids two to three days before a scheduled screening session on the Thermo Scientific Talos™ Arctica. There is a maximum of 11 grids.
- With our assistance, clip your grids into autogrid cartridges that are compatible with Thermo Scientific Talos™ Arctica and Thermo Scientific Titan/Krios™ G3i.
On the screening day, the typical workflow is as follows:
- We insert your grids (maximum of 11) into the Thermo Scientific Talos™ Arctica.
- For each grid, we collect a whole-grid montage at low magnification, several grid square montages at intermediate magnification, and several high-resolution images. These images allow you to evaluate whether the electron microscopy grid is suitable for high-resolution imaging.
- We save suitable grids for high-resolution imaging at a future date with the Thermo Scientific Titan/Krios™ G3i, or we can collect data overnight with the Thermo Scientific Talos™ Arctica. Although the latter produces slightly lower-resolution images, two-dimensional classification would indicate whether a desired complex is present or whether a sufficient variety of views are available to allow for three-dimensional reconstruction.
- We can automate overnight data collection if your grid has a regular hole pattern. If you would like to manually select specific locations on the grid, you must be present to guide the selection process before we begin collecting data.
- During data collection, dedicated computer servers perform motion correction and contrast transfer function determination of the images, which are typically collected at the rate of approximately 45 per hour. The results of these preliminary analyses are ported to a website that you can monitor on a real-time basis.
- For internal users, these data are uploaded to NYU Langone’s terascale high performance computing cluster, Phoenix, where you can begin the process of two-dimensional classification and three-dimensional structure determination using, for instance, RELION or cryoSPARC.
You are encouraged to provide your own electron microscopy grids according to the specifications outlined below. The Cryo-Electron Microscopy Laboratory has a limited supply of grids and storage boxes available for purchase to get you started on a project. If you need assistance choosing the appropriate grids or wish to discuss purchase of supplies, please contact Bill Rice, PhD, director, at firstname.lastname@example.org or Bing Wang, PhD, research scientist, at email@example.com to discuss how we can best assist you.
Electron Microscopy Grids
For single particles, you will need to obtain grids that are coated with a carbon film containing holes. The two major brands are QUANTIFOIL® and C-flat™. QUANTIFOIL® grids are more commonly used, but C-flat™ grids are preferred by some researchers because they have a thinner carbon film without a lip at the edge of each hole. The claim is that this helps to produce a thinner layer of ice.
Grids are available with various hole patterns, as summarized for QUANTIFOIL® in the table below. To start, we recommend you use R 2/2 Cu grids.
|Style||Hole Diameter||Space Between Holes||Center-to-Center Distance|
|R 2/1||2 µm||1 µm||3 µm|
|R 2/2||2 µm||2 µm||4 µm|
|R 2/4||2 µm||4 µm||6 µm|
|R 1.2/1.3||1.2 µm||1.3 µm||2.5 µm|
|3.5/1||3.5 µm||1 µm||4.5 µm|
|R 1/4||1 µm||4 µm||5 µm|
|R 5/20||5 µm||20 µm||25 µm|
|R 0.6/1||0.6 µm||1 µm||1.6 µm|