The ThermoFisher Krios Cryo-TEM offers a stable 300kV cryogenic platform designed for single particle analysis and tomography. This is accomplished by constant power to the lenses that will limit linear distortions and reduce thermal drift. It has a 12 grid autoloader that allows for batch screening of samples. The dalta collection software permits the acquisition of large image sets automatically. The Duke SMIF Krios Cryo-TEM is configured with the following:
- Gatan K3 Bioquantum (K3 + energy filter)
- ThermoFisher Falcon2
- ThermoFisher Ceta 16M Camera
Data Collection Software
- ThermoFisher EPU
- Gatan Latitude
Standard Krios Data Collection Conditions
If you would like to use data collection conditions that are different than those shown below, you will need to contact SMIF staff and request a discussion of the proposed change in writing at least one week in advance of your scheduled session. These changes must be approved by the SMIF staff in writing before Krios usage.
Single Particle Data Collection
- Detector used: K3
- Software used: Latitude
- Energy Filter: Not used*
- Data Collection Magnifications: 81,000x, 105,000x
- Hole-to-hole imaging: Beam tilt
*The energy filter is currently not always functioning properly and Gatan is coming to address the issue. Once it is fixed we expect to add usage of the energy filter as an option under the standard conditions.
SMIF Krios Reservation and Usage Policies
- Users utilize Core Research to make a reservation request (see link above for "How to Make a Reservation Request in CoreResearch").
- Reservation approval by SMIF staff is required.
- Users must indicate the following in the reservation notes:
- Project/sample approval code (from the Krios Project Approval Process).
- Identification of the individual who is going to load/run sample (SMIF Staff or approved Super User).
- Email address and contact telephone number for the user (which should accept texts).
- (Optional: instructions regarding the Express queue).
- SMIF staff assistance can be requested when making a reservation.
- Reservation approval by SMIF staff is required.
- Time is reserved in 24 hour timeslots, with a minimum reservation of 24 hours and a maximum reservation of 96 hours in one week (Monday morning to Monday morning).
- Reservations are not allowed to start or end on a Saturday or Sunday, without prior SMIF approval.
- Each approved project may have up to a maximum of 2 active reservations on the Core Research calendar, with no more than 4 consecutive calendar days reserved, and no more than 4 active reservations per PI/Lab Once one of these reservations has been completed or cancelled, additional reservations can be made as long as the limits on total number of active reservations are kept.
- Reservations must be made at least 1 week in advance of the reservation start and can be made up to 2months in advance of the reservation start.
- Cryo-Cycles are scheduled to be run overnight on Thursday every two weeks, and these are pre-populated on the CoreResearch calendar. Reservations should be made for the time durations shown below in order to optimize Krios usage:
- Weeks without a Cryo-Cycke
- Mon AM-Fri AM (4 days) or Mon AM-Wed AM (2 days) and Wed AM-Fri AM (2 days)
- Fri AM-Mon AM (3 days)
- Weeks with a Cryo-Cycle
- Mon AM-Thurs 3pm (3.5 days)
- Fri AM-Mon AM (3 days)
- Reservations may be cancelled by the user up to 3 business days in advance of the reservation start with no charge.
- If the sample associated with the reservation is unusable (e.g. unavailable, damaged, etc.) at the start of the reservation time, or if a session is aborted due to sample problems, the user will be charged for any unused reserved time that is not utilized by another user.
- Documented instrument failure will not result in usage charges.
- Two types of samples will be stored in the Krios dewar for imaging:
- Unclipped stored in a grid box, with a hardcopy grid map.
- Clipped grids stored in an autoloader grid box. Documentation for clipped grids must include photos of the grids and a hardcopy grid box map.
- Grids will always be inspected by the individual performing the loading before the sample is loaded. If the grid inspection reveals a grid that does not match the grid photo, the person loading will take a second photo. If the grids are damaged to the extent that the autoloader operation may be compromised, they will not be placed in the Duke Krios and the user will be contacted. Assessment of damage will be by SMIF staff or the Super User loading the grids. Damaged grids will be placed back in the Krios dewar. Typically, multiple samples from multiple projects will be loaded into the autoloader simultaneously (typically up to 3 samples per project and up to 9 grids total).
- Users are encouraged to place samples into the Krios dewar at least one week in advance of the imaging reservation. Users should be available for questions via telephone or in person when the samples are scheduled to be loaded and for 2 hours after loading. If the SMIF Staff or Super-User who is loading the Krios have concerns about the samples when loading, and the user cannot be reached, the grids will not be placed in the Krios, and will be placed back into the Krios dewar.
- Samples must be located in the SMIF Krios storage dewar at least one business day (24 hours) for clipped grids or two business days (48 hours) for unclipped grids prior to the start of the reservation period.
- Users are encouraged to submit samples at least one week in advance to facilitate loading of multiple projects and to enable access to the Express queue.
- If samples are not in the SMIF Krios dewar one business day (24 hours) for clipped grids or two business days (48 hours) for unclipped grids, then SMIF staff may cancel the reservation. The user will be charged for the reservation time unless another user utilizes the reservation time.
- Sample loading will generally be planned according to the following run schedule:
- Runs of 24-96 hours: Monday AM – Friday AM; anticipated loading Mon morning;
- Runs of 72-96 hours: Fri AM – Monday AM; anticipated loading Friday morning.
- Weekend guidance: Loading of samples is permitted on weekends by Super Users and SMIF Staff, with SMIF Staff prior approval. Express samples are automatically pre-approved by the SMIF Staff.
- All samples imaged in the Krios must be associated with a Core Research reservation.
- Any samples placed in the SMIF Krios dewar one week or more (limit 3 weeks) ahead of a scheduled reservation will be placed into the Express queue unless the user specifies in writing in the reservation notes that it should not be placed in the Express queue.
- Samples in the Express queue will be run on a first come basis if time opens up on the Krios prior to the scheduled reservation for that sample. If a sample is being run as an Express sample, the individual loading the sample into the Krios (SMIF Staff of Super User) will text or call the user at the telephone number in the reservation notes before the sample is loaded. Express users are encouraged, but not required, to be available by telephone with the individual loading and running the sample when the sample is loaded, and for 2 hours after loading.
- When Express samples are run prior to their originally scheduled reservation, the original reservation is then made available to the user who made the reservation on a first refusal basis. If that user does not use the original reservation, then any other user can take that (now open) reservation.
- Samples in the Express queue that are not run earlier than scheduled will be run during their originally scheduled reservation.
- Samples may not be placed in the SMIF Krios dewar more than 3 weeks prior to their scheduled reservation.
- Cryo TEM for single particle analysis
- 12 Grid Autoloader
- Gatan K3 Detector
- Falcon2 Detector
- Volta Phase Plate Integration
- Automated Alignment Routines
- 1.8Å resolution