Policies
Usage and Billing
For any new projects, the Project Proposal Form must be filled out and signed. A copy of the form will be emailed to the facility managers, user and their PI. For any biohazardous samples, the protocol must first be approved by the Biosafety committee. A copy of the biosafety protocol must be forwarded to the Managing Director.
Reservations
Independent users may sign up for microscope time. Trainees must check with the Managing Director for available time, or only shadow independent users in the session.
Session Requirements
Due to the high number of images necessary for a single particle reconstruction and a tomographic exploration, the minimum sign-up time for the Arctica and the Krios is 1 day. Each one-day session is from 9 am to 9 am on the following day. Users are required to remove their grids by 9 am to allow for a smooth transition for the next user. Or if they agree, the next user may remove the grids for them. The maximum session length on the Arctica and the Krios is 2 days. Under extenuating circumstances (very heterogeneous sample, etc.), longer sessions can also be approved. Please contact staff to discuss the possibilities. To allow for equal access to the facility's resources, a user can only sign up for a single session at a time. Reservations can only be made up to a month ahead of the current date. Any cancellations must be made at least 24 hours in advance of the session.
Every Monday on the Arctica is designated as a "Flex" day. The primary uses for this day are 1) for emergency collection or to make up a collection day due to unexpected microscope downtime and 2) as a screening day. For screening, two 4-hour sessions can be reserved: 9 am – 1 pm, and 1 pm – 5 pm. Independent users could set up an overnight collection from 5 pm to 9 am the following morning. Please be prompt in removing your grids Monday morning before 9 am and/or at the end of your screening session. The sessions on Mondays will not contribute to your reservation count.
On any day when nobody signs up on the Arctica or the Krios, user can come in and use the microscope for any amount of time, until 9am the following morning. The session will be billed for actual time used, rounded to hours.
It is expected that before single particle project users sign up for time on the Arctica, they will optimize new samples through negative stain imaging on the T12 until the particles are (1) monodisperse, (2) abundant, and (3) classify into nice 2D classes (evidence of structural stability). Though due to the resolution limit from low coherence thermionic gun on the T12, the 3rd criteria might not be met before moving on to cryo. All cryo-sample optimization should be done on the Arctica, including at least one full reconstruction. This step will reveal whether the particles resolve into clear class averages (hopefully with visible secondary structure) AND whether orientation preference is a problem.
Tomography samples should be optimized on Arctica until the (1) target abundance, (2) ice quality, and (3) number of fiducials per field of view are all suitable. Cryo-FIB'ed lamella, cryosections, and grids that have already been imaged by cryo-fluorescence microscopy can all go directly to the Krios.
Biosafety
Every user, regardless of the hazard level of their sample, should be prepared to discuss:
- Preliminary risk classification/assessment for the material they are bringing
- Best cleaning/disposal practices
- Best handling practices
- Minimum infectious and lethal doses
- For infectious agents:
- Rates of survival on various surfaces
- Preferred areas to infect
- Transmission source/routes of exposure
- Types of infections caused
- Symptoms and incubation time
- Types of treatment available
This information and any specific procedures necessary for the safe handling of the sample must also be provided to facility managers.
Consumables
The Center maintains all cryo-EM tools and supplies except for grids. Due to many options for kinds of grids and necessary careful storage and handling, individual labs must plan in advance and provide their own grids. Individual labs are also welcome to use their own tools, e.g. the vitrobot tweezers, for possibly better tractability. Most consumables are free except for the autogrid C-clips and C-clip rings that can be ordered from Idea Elan's "Supplies" section.
Downtime
Due to the complexities of the instruments, user error, or unforeseen circumstances unplanned outages do occur, potentially interrupting the user's session. When this occurs, the billed usage will be rounded to the nearest hour. Further, due to the complexities of trying to shuffle everyone when outages occur, any user with an interrupted session would have to sign up for a new session at the next available time.
Sample Storage Policies
The cryo-EM facility is a shared space open to all Caltech researchers. Each user must return shared resources to their designated location as soon as possible. We can provide a short-term space in the 4°C and -80°C fridges and freezers, as well as cryo specimen dewars. Each user trained in freezing will be allocated a single puck (12 grid boxes) for sample storage. Shared spaces will be regularly cleaned out and it is the user's responsibility to adequately label and store samples and reagents in order to prevent any accidental discharge of the material during cleaning.
Acknowledgments
Please provide the following statement in any publications that used center resources:
"(Cryo) Electron microscopy was done in the Beckman Institute Resource Center for Transmission Electron Microscopy at Caltech. "
Please send citations to any paper that utilized center resources to facility managers as soon as it is published.
Revoking Independent User Status
The Independent User status and associated privileges (independent sign-ups, weekend sessions) can be revoked at any time if the user is observed not to be following established SOPs or microscopes require repeated maintenance after the user's session. The status could be reinstated after additional training sessions and the display of correct protocols by the user. If the independent users haven't use the facility for more than 6 months, they automatically lose the status and will need re-training to get it back.