Our electron microscopes, especially the Titan Krios Cryo-EM, are very expensive and delicate instruments, with a high level of demand. Everyone desires greater access, high sample throughput and superior results. So, access is regulated. Moreover, it is sample quality and not the microscope that impacts throughput and results most often. We strongly suggest that the sample quality and screening protocols below be followed.
Titan Krios Cryo-EM Access & Use
- Submit the Project Registration Form if not completed. At this time, projects are not being reviewed and approved. The purpose of registration is to aid the upcoming instrument scheduling, fee recovery and infrastructure development.
- Use of this microscope requires an appropriate degree of individual training, as it can be easily misaligned, misconfigured or damaged by improper use. While Cryo-EM facilities at other institutions can require multiple years of experience (e.g. 4-5 yrs) from users to fly solo on a Titan Krios, we will qualify users more quickly as Super-Users when EM staff decide an individual has shown proven ability. Fill out a training request to schedule a demonstration of your knowledge and skills with the Titan Krios Cryo-EM.
- Only Staff and Super-Users are allowed to align the electron beam unsupervised. Reiterating, you must become a Super-User in name to forego supervision during microscope operation, even if you have already been trained to do certain tasks and remember how to this and other things. Lab PIs of any users that break this rule will be charged for the staff time required to get the microscope operational. Again, first make a training request to demonstrate knowledge and skills when you are ready.
Macromolecular Sample Quality Controls
- Samples should be chemically and structurally homogeneous, as well as functional, for the majority of project aims envisioned (e.g. single SDS-PAGE bands, pooling only HPLC fractions above the half-height maximum of single chromatogram peaks avoiding any shoulders caused by other species, avoiding final sample over-concentration or any other causes of aggregation, etc). While this preliminary data will be required only for projects competing for our mini-grants and vouchers at this time, such data is key to avoid wasting lots of effort and paying for bad results. Because sample quality is so important, a separate section titled Sample Preparation contains more information and supporting figures.
- The sample quality following application to grid surfaces should be quickly screened by transmission electron microscopy (TEM) using uranyl salts to provide particle contrast. Here, this step uses our BioSpirit electron microscope. You want to see an absence of aggregates and contaminates at lower magnification and a suitable particle distribution as a function of sample concentration at higher magnification.
Cryo-EM Grid Quality Controls & Screening
- Presently, the Titan Krios Cryo-EM should be utilized to screen frozen samples applied to EM grids to validate the following conditions: a) Vitrobot parameters for a thin vitreous ice, b) the best sample concentrations and buffer components that allow numerous monodispersed particles to be found inside grid holes, c) enough grids with sufficient grid squares with said particles in thin ice for storage and a successful data collection, and d) your levels of luck and ingenuity. Off site users may instead opt to use a screening Cryo-EM located at their own institutions, as this optimization step can take awhile to complete. If one observes two grids out of one batch appear optimized, then place the remainder unscreened into LN2 storage as moving grids in and out of cryo-microscopes is itself a dangerous step, as quite often a layer of ice crystal frost will coat these more manipulated grids.
Shipping Cryo-EM Grids Here
- External users collaborating with EM staff members or paying for data collection as a service must ship their screened frozen EM grids to The Hormel Institute address, C/O Cryo-EM Lab Staff. Please ensure that your samples arrive 1-4 days prior to the scheduled data collection and communicate with EM staff members before you ship so proper LN2 storage can be insured upon arrival. You are also welcome to come here along with your samples, to receive training and/or to be present during data collection.
Titan Krios Cryo-EM Fees
- When Titan Krio time is allocated in daily slots, we charge a daily rate. Whenever instrument time is allocated in hours, an hourly rate is charged. Hourly use is only envisioned for grid screening and perhaps for some Cryo-ET at the present time. We believe our fees are below that of nearly all similar Cryo-EM facilities, except those that offer some limited form of free access. We also offer “free access” in the form of microgrants and voucher awards to suitable competitive projects. Super-Users usage is charged a lesser rate since they do not require as much staff effort and mentorship. The fees for External Academic and Commercial use are more, but they remain highly competitive in comparison to all other Cryo-EM facilities throughout North America. If a scheduled data collection must be stopped due to any problems that develop within our instrument(s) or infrastructure, you will only be charged up until the time that the problem developed, pro-rating the fee as required. It is most likely your grids will still be safe and ready once we are operational again. However, we do not discount the daily rate upon learning you only had poor quality samples that did not provide the results you desired.
Processing Computation, Software and File Storage for Titan Krios Cryo-EM Data
In addition to local workstations, the EM Labs relies on the excellent computational facilities that The Hormel Institute IT department maintains: a dedicated CRAC unit, a 300 kVA UPS backup system, a backup tape library, and six racks of compute and storage server systems, which are actively undergoing updating and improvement.
- To collect data on the Titan Krios G2, you will use ThermoFisher Scientific’s software, Atlas and EPU. However, Gatan’s Lattitude software will soon be implemented when the Biocontinuum Detector upgrade is complete replacing EPU.
- The current Titan Krios G2 Cryo-EM produces ≥10 TB of data every 2-3 days on two file storage systems. A pool of 70 TB of disks is attached directly to a storage server connected by 10 GbE fiber to the microscope’s acquisition system. Data from the microscope’s server are then rapidly replicated onto a 570 TB Distributed Data Network (ddn) parallel storage system via GPFS-CIFS protocol nodes for initial data processing and implementation of backup image storage. From here, you may access your data for a period of roughly 2-3 weeks at no additional cost. While there is presently no period set for data removal from this temporary data processing space, users are expected to move data files onto their own file systems quick enough that other’s on-going data collections are never impeded. Instead of implementing a data bomb period, we will attempt to contact the owners of stale files that have already been backed-up whenever storage space emergencies occur. However, we reserve our right to remove long-resident files from the ddn system.
- The IT department presently maintains Cryo-EM image backups at no cost to users of the EM Labs. Because ddn disk utilization consistently remains very high, a very large 3 PB SpectraLogic T950 Tape library and Blackpearl high performance image storage system with one tape frame and an online capacity of over 3 PB (1 PB = one million gigabytes) was deployed in 2020. Without additional tape library frames, the current system can be expanded to roughly 8 PB using LTO-8 tape media. All backups presently use dual tape copies to provide extra security and thus the usable capacity is one-half of the total online capacity. Since funding for a Titan Krios G4 and a third screening Cryo-EM has been identified, another 3 PB image storage system is also soon planned for installation.
- A vxRail system with VMware HCI software comprises our computing infrastructure, a cluster that is constantly being expanded towards a maximum of 64 nodes. In 2020, nodes were deployed featuring the newest AMD EPYC and Intel Processors and high-end NVIDIA GPU Computing. Four new nodes each with four Nvidia RTX 6000 GPUs were added in January 2021. Additional nodes and GPUs were then added in February 2022, and these upgrades will continue to keep pace with demands. In addition to this new hardware, interactive computing software (NICE/Enginframe) is employed to meet our researcher’s interactive computing needs. Finally, The Hormel Institute’s WI-FI and network were recently modernized, firewalls were implemented, and several additional storage and computer systems were updated.