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Policies and Guidelines for Cryo-Electron Microscopy at CEMAS


Project Qualification

All users interested in using the cryo-electron microscopy facilities at CEMAS must first complete the Cryo-EM Project Form (http://go.osu.edu/cryoemproject). The form will be reviewed by CEMAS staff to ensure sample(s) qualify for imaging.

Reservations and Allocation

Users who can provide evidence that their samples are of acceptable quality will be given priority access to the instrument on a first-come, first-served basis.

All reservations are made using the Facility Online Manager (FOM) site (https://fom.osu.edu) with a valid account.

To allow for equal access to the cryo-EM facilities, at any one time, a user may have 1) one screening session and 2) one data collection session. Once the sessions have been completed, the user may sign up for another reservation.

Reservations can be made up to one month ahead of the current date.

  • To ensure maximum utilization of the Krios, if a reservation is still open one week ahead of time, it may become available to all users who have access to the Krios, including those samples that might not be qualified. If a user chooses to utilize this Krios capacity and currently holds a Glacios data collection reservation, the user must cancel the Glacios reservation to free up space for other Glacios users.
  • Any reservation that is open 24 hours ahead of time may become available to all Krios users without counting toward the reservation limit. Krios users must contact the Instrument Manager to schedule.

Sessions

Category I: Conventional cryo-TEM imaging
Glacios sessions are hourly

Category II: Single particle analysis 
Session minimum: the minimum sign up time for the Glacios and Krios is one day (24 hours). 
Session maximum: the maximum session for the Glacios and the Krios is two days (48 hours).

Cancellations

  • Cancellations made at least 48 hours in advance of the time slot start time will not be charged a cancellation fee. 
  • Cancellations less than 48 hours in advance of the reservation time will be responsible for the costs of the reservation, unless another user takes the released reservation.
  • When a session is cancelled within 48 hours, an email will be sent from the FOM system to users who have instrument access, notifying them of the availability.

Sample and Grid Handling and Tracking

Safety

  • Regardless of hazard level of the sample, every user should be prepared to discuss the risk classification for the material of the sample.
  • Hazardous material cannot be brought into CEMAS (even if frozen) without prior approval from CEMAS staff.

Sample Storage

  • Due to limited space, users should not store their samples, solutions, or other materials unless authorized by CEMAS staff. 
  • Short term storage in the form of a small 4° C fridge is available in the Bio Prep Lab (room 122) to temporarily store samples.
  • There will be long term storage available for frozen grids.

Sample Preparation

 

Consumables

Consumables, such as grids, are available from CEMAS at cost.

Microscope Operation

Thermo Scientific Glacios

The Thermo Scientific Glacios can be used for both cryo-sample screening and cryo-data collection.

Thermo Scientific Krios

The Thermo Scientific Krios can only be used for cryo-data collection of Krios qualified samples.

Prior to collection on the Krios, grids must be screened on the Glacios to optimize collection time. For single particle analysis, a preliminary reconstruction may be required from a previous collection.

Operation

Due to the difficulty and high chance of errors that can take a microscope down for extended periods of time, at this time all operation is to be performed by CEMAS staff only. This includes all grid clipping, loading, unloading, transfers, microscope alignments, low dose imaging and data collection, and troubleshooting. Under rare circumstances, CEMAS staff may permit qualified users to perform some of these tasks independently.

Unforeseen Microscope Interruptions

The cryo-EM instrumentation is very complex. Unforeseen circumstances could potentially interrupt a user’s session. CEMAS staff will do their best to minimize interruptions within their control. However, if a defect does occur, the billed usage will be rounded to the nearest half-day.

Users with an interrupted session will need to sign up for a new session at the next available time due to the complexities of rescheduling later sessions.

Microscope Performance and Quality Checks

Cryo-Cycle

A cryo-cycle is a critical form of maintenance which includes a warmup cycle to expel water that has built up on the cold surfaces. Cryo-cycling of the microscopes will be performed once a week for 24 hours to avoid buildup of contamination. 

Data Processing

At this time, users are required to perform their own data processing and analysis. 

Training and Access

Training for the use of electron microscope instrument will be carries out by the facility manager, who is authorized by the CEMAS Director to decide when and whether a trainee has sufficient knowledge and proficiency to be designated an advanced or expert user. Equipment training may be requested through the Facility Online Manager (FOM) site.

  • basic user is one that must work on the microscope with the facility manager or other CEMAS staff member present. This type of user can only work during the hours of 9am to 5pm, Monday through Friday. Any overnight data collection runs must be ready by 5 pm. Sample preparation can be completed anytime during regular business hours without supervision.
  • An advanced user is one that can work independently from the facility manager, meaning the user is certified to work weekends, holidays, and overnight when the facility manager is not present. For certification, individual users must demonstrate proficiency in performing all required steps and tasks independently, including but not limited to items 1-7, listed below. Advanced users should be able to perform all necessary steps without having to refer to their notes, and while operating all instrument components safely.
  • An expert user has the same access as an advanced user and may be qualified to load the cassettes into the microscope. Expert users are frequent and regular users of both the Glacios and Krios microscopes.

 

Tasks

Basic

Advanced

Expert

1. Sample preparation on the Vitrobot

2. Grid clipping

3. Grid loading into cassette for Nanocab

4. Microscope operation without facility manager (after-hours access)

5. Microscope alignment

6. Setup EPU for long data collection – low dose imaging

7. Basic troubleshooting

A detailed explanation of the tasks listed in the above table is given below. These are only guidelines and may be expanded, if necessary, to ensure safe microscope operation.:

Sample Preparation (#1):

All users (basicadvanced, and expert) of the Vitrobot must be able to demonstrate the following:

  • Correct setup of the instrument including:
    • How to handle and produce liquid ethane
    • Produce ice of the correct thickness and appropriate sample concentration
    • Properly freeze samples with minimal ice contamination
    • Follows the correct shutdown procedure, leaving the instrument properly stored (door open, blotting pads and tweezers removed, water container emptied)
  • Ability to handle grids with minimal damage or loss of TEM grids
  • Understanding which grids are appropriate to use for different applications
Grid Clipping (#2):

All advanced and expert users must be able to show:

  • How to assemble the clipping station
  • How to properly load a c-clip
  • Test if the clipping was done correctly, what factors to consider when placing the grid inside of the ring (bent grids, etc.), how can you tell if a grid has been clipped?
  • What precautions should be taken to prevent ice contamination?
Grid Loading (#3):

Only a small number of specially trained expert users will be permitted to fill the cassette for loading into the Autoloader.

Microscope Operation (#4-5)

All advanced and expert users must be able to perform direct alignment procedures:

  • Adjust pivot points
  • Center C2 aperture
  • Fix rotation center
  • Coma-free alignment
  • Set conditions for parallel illumination in diffraction mode.
Setting up EPU (#6):

All advanced and expert users must demonstrate proficiency in the use of EPU. This includes:

  • How to decide which EPU settings to import; how to export new EPU settings
  • Properly collect an Atlas; how to import previously collected Atlas images
  • How to start and save an EPU session
  • Add squares for data collection
  • Demonstrate use of the Autofunctions: Autoeucentric and Autofocus
  • How to find and add/subtract holes for data collection
  • Correcting for the objective stigmation; Check the FFT during data collection
  • Explain differences in setup procedures for Quantifoils versus Lacey Carbon grids
  • What to do if EPU freezes?
Troubleshooting (#7):

Advanced and expert users are expected to know how to address the most common errors:

  • Ice is too thick
  • Grid has too much damage/ice contamination
  • Sample is too concentrated or too dilute
  • Forget to change the exposure time on the Data Acquisition tab
  • Forget to click “Get” after finding the parallel illumination conditions
  • Forget to set the eucentric height for EACH grid square independently
  • Common EPU error messages – e.g., Too high of an exposure on the Falcon
  • Adjust exposure time after measuring dose

Most importantly, users must be able to assess what does/does not constitute basic troubleshooting vs problems that require immediate notification of the facility manager.