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.

Workflow

The typical workflow is as follows:

• Obtain or prepare a sample to image, for instance, a column-purified solution of a macromolecular complex. Ideal concentration range is ~10 µM, sample dependent.
• Optionally, check this sample by negative-stain electron microscopy using, for example, Thermo Scientific Talos™ L120C microscope in the Microscopy Laboratory. About 1/10 concentration is needed for negative stain compared to that needed for cryo.
• Freeze electron microscopy grids containing your sample using our Thermo Scientific Vitrobot™ Mark IV, Leica EM GP, or chameleon. For blotted grids, 3-4 µl per grid are required. For chameleon, 8 µl total is required. We can advise or help you to prepare samples. A variety of freezing conditions need to be explored for new samples.
• Store your grids in our liquid nitrogen dewars.
• Clip your grids into autogrid cartridges that are compatible with Thermo Scientific Talos™ Arctica and Thermo Scientific Titan/Krios™ G3i. We can clip or train you how to clip. Autogrid clips and rings are available for purchase.
• Book a screening day on the Arctica to check your samples.

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 atlas (montage) at low magnification, several grid square images 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.
• Leginon is used for screening and collection. It has an excellent automated holefinder, the ability to select ice thickness ranges for imaging, and a clean, logical workflow and data storage organization. All metadata is automatically stored in a database and accessible via a web page.
• Frame alignment and CTF determination are set up to happen automatically during collection and images can be seen on the Leginon website. We run Warp particle picking live so particles are picked as soon as they are collected. 2D classification is done in batches using cryoSPARC and/or Relion.
• Generally, at least 10,000 particle picks are needed for 2D class averages. For 3D reconstruction, we like to have a to least 50,000 initial picks.
• For screening, we prefer to choose a wide range of ice thicknesses. For further collection, we can specify the ice thickness range depending on where the particles lie.
• After the initial screen, we can optionally image the best grid to the end of the session. The session can be extended to overnight collection if desired.
• 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.
• Fully automated collection happens at about 250 images/hour on the Arctica and up to 350 images/hour on Titan Krios. For an overnight Arctica collection, expect 3000+ images. For a 24-hour Krios collection, expect 6,000-8,500 depending on grid quality.
• All data are uploaded to NYU Langone’s high performance computing cluster, UltraViolet. Internal users will have automatic access to the data as it is collected. External data is usually shared through a GLOBUS server.
• For screens of more than 12 grids, Smart Leginon will be used.

On the Tuesday fast screening day, typical workflow is as follows:

• We insert all samples (up to 11 grids) in the morning
• Up to 4 grids can be screened for 2h, charged by the grid.
• During your session, we screen through your grids to get an idea of overall quality, ice thickness and particle count. Due to the short time, expect 100-200 images maximum.
• One session from the day can go overnight, if desired. First to ask gets the overnight, which is charged a half-day session

For the Titan Krios collections, typical workflow is as follows:

• Load the best prescreened grid, collect an atlas, and set up imaging as soon as possible.
• Optimize the ice thickness settings as collection continues.
• Change grids as necessary.

Supplies

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 William Rice, PhD, director, at William.Rice@NYULangone.org, Bing Wang, PhD, senior scientist, at Bing.Wang@NYULangone.org 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.

The most common grid types are Cu and Au. Au grids are required if cells are grown on grids since Cu is toxic. Au grids are softer and easier to bend.

Foil types available are carbon and Au. In general, it is easier to get thinner ice on Au foil grids. Additionally, there is less beam induced specimen motion on Au grids. Au grids are strongly preferred for tilted data collection. Using our SafeMatic evaporator and Gatan Solarus, we can make our own Au foil grids from carbon grids. These grids work just as well as commercial Au foil grids without needing to wait months for them to be in stock.

Grids with thin carbon or graphene oxide over holes are available for samples which are difficult in pure ice. Let us know if you want to try them.

QUANTIFOIL^® grids and C-flat^™ grids can be purchased from Electron Microscopy Sciences, Ted Pella or directly from Quantifoil.

Grids are available with various hole patterns, as summarized for QUANTIFOIL^® in the table below. To start, we recommend you use R 1.2/1.3 Cu grids.

QUANTIFOIL® Grid Hole Patterns

Style	Hole Diameter	Space Between Holes	Center-to-Center Distance
HexAuFoil	0.33 µm	0.32 µm	0.65 µm
R 0.6/1	0.6 µm	1 µm	1.6 µm
R 1.2/1.3	1.2 µm	1.3 µm	2.5 µm
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
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