Preparing Samples, Data Collection and Processing
Researchers performing cryo-EM experiments can acquire the training to make their own cryo-EM and negative-stain grids, and collect and process their own high-resolution data. We also offer sample preparation, data collection and data processing services. Users have access to two transmission electron microscopes. Core staff members are available to consult during project development.
To request services or training, please refer to our rates and scheduling page.
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Schedule With Us
To schedule cryo-EM services with the Electron Microscopy shared resource, or to get more information about how we can work with you, contact our EMSR team:
Theo Humphreys
Director, Electron Microscopy
Sample Preparation
We are equipped with standard sample-preparation instruments used in negative stain and cryogenic electron microscopy grid generation. Our team provides sample preparation services as well as training in these techniques. We consult with users looking to optimize freezing conditions and overcome challenges, such as preferred orientation and low particle density.
Training
We provide structured training for both newcomers and users with previous experience in single-particle electron microscopy analysis. We review electron microscope safety, negative staining, carbon coating and cryo-EM grid-freezing protocols in a one-on-one format. Users can schedule troubleshooting consultations with core staff as needed. We grant users independent-use status and door access to the cryo-EM laboratory space after they pass proficiency tests at the end of each training section.
Users have access to:
- Two-part negative stain bench training
- Three-part Vitrobot training
We provide negative staining services for single-particle samples provided by users. Sample handoff must be scheduled at least 48 hours in advance. Users must provide samples and buffers.
We provide cryo-EM grid-freezing services for single-particle samples provided by users. Sample handoff must be scheduled at least 48 hours in advance. Users must provide samples and buffers.
Our staff will clip and load autogrids for use on the Glacios cryo-EM transmission electron microscope. Grid handoff must be scheduled at least 24 hours in advance.
This thin-film deposition system provides full-range, low-voltage thermal carbon evaporation and can produce fine-grained thin films via sputtering.
Related service: Thin carbon coating of grids
This instrument supports automated grid vitrification in liquid ethane. Users must provide their own samples and grids; we will provide all other necessary materials and tools.
Related service: Cryo-EM grid freezing
Data Collection and Processing
Our core aims to empower users to collect and process their own cryo-EM data. We train users to operate two different transmission electron microscopes, the Talos L120C and the Glacios. Users will learn how to screen grids and collect high-resolution cryogenic data, as well as how to process their data using facility computational resources.
The core provides access to single-tilt grid holders for room-temperature and cryogenic applications using the Talos microscope.
Temporary data storage is available for one month after data collection. On-the-fly motion correction and pre-processing are provided with Glacios reservations. We encourage users to consult with core staff during all stages of project development.
Training
We provide structured training in single-particle electron microscopy analysis for both experienced and new users. Core staff will work with researchers to set up negative-stain data collection and cryogenic screening on the Talos L120C microscope. We offer training in cryo-EM screening and large-dataset collection for single-particle projects on the Glacios microscope.
We will grant users independent-use status and door access to the microscope rooms after they pass proficiency tests at the end of each training section.
Our staff will collect a full dataset on prepared negative stain grids using data-collection parameters decided during consultation with users. We will provide micrographs to users immediately following collection.
Our staff will collect and process negative-stain data up to the point of generating 2D class averages to assess particle size, morphology and homogeneity. We will provide class averages less than four business days after we complete collection.
Users are welcome to contact us with any processing questions, office hours are available upon request.
Please reach out to us at emsr@fredhutch.org.
These resources may help shared resource users expand their cryo-EM knowledge:
Cryo-EM
- Caltech course: Getting started in cryo-EM
- Cryo-EM: A Unique Tool for the Visualization of Macromolecular Complexity
- Cryo-EM maps are full of potential
- Present and Emerging Methodologies in Cryo-EM Single-Particle Analysis
Leginon
- MSI operation wiki
- Leginon: A system for fully automated acquisition of 1000 electron micrographs a day
- Leginon: New features and applications
SerialEM
RELION
- A Bayesian view on cryo-EM structure determination
- New tools for automated high-resolution cryo-EM structure determination in RELION-3
- Single-particle processing in RELION-3.1 PDF
- Getting started with RELION
cisTEM
cryoSPARC
- cryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination
- Non-uniform refinement: Adaptive regularization improves single-particle cryo-EM reconstruction
- 3D variability analysis: Resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM
- Cryo-EM data processing in cryoSPARC: Introductory tutorial
Equipment
Please see Talos L120C in TEM Equipment for microscope details. Click here to access.
Trained users will have access to:
- Single-tilt room temperature holder
- Elsa cryo-transfer holder
- TEM Imaging & Analysis (TIA) data-collection software
- Velox data-collection software
- Leginon automated data collection software
- Database of all user-specific metadata collected using the Leginon via the Hutch Leginon webserver
Related services: Negative-stain data collection and two-dimensional averages.
This TEM from Thermo Fisher Scientific is operated at an accelerating voltage of 200 kV and is equipped with a 24 megapixel (5,760 x 4,092) Gatan K3 direct electron detector which can record data at 1,500 frames per second. The Glacios is used for screening and high-resolution data collection of cryo-EM samples. The 12-grid cryo-autoloader allows for efficient and reliable screening of multiple samples and the XFEG electron source offers a bright and coherent electron beam for high-resolution images.
Users will have access to:
- 12-grid autoloader
- TEM Imaging & Analysis (TIA) data-collection software
- Leginon automated collection software for single-particle data collection
- SerialEM automated collection software for data collection
- On-the-fly drift correction and processing for the duration of the microscope reservation
- Data storage for 30 days post-collection
- Database of all user-specific metadata collected using Leginon via the Hutch Leginon webserver
Available for users during their data collection sessions on the Glacios/K3, this machine runs Warp EM for on-the-fly motion correction and preprocessing, including contrast transfer function (CTF) estimation and particle picking.
Server specs:
- Dual Xeon Gold 6254 3.1 GHz 18-core CPU
- 4x PNY NVIDIA RTX A6000 GPU
- 384 GB RAM
This storage server will be available for one month of temporary data storage for all data generated during a data-collection session, including raw movies and aligned micrographs.
Server specs:
- Dual Xeon Gold 6248 2.50 GHz 20-core CPU
- 648 TB HDD storage
- 10-GbE connection
These 4 GPU workstations, Nocturne and Etude, are preloaded with standard cryo-EM data processing sofrware and can access users' data as it is being collected. Workstation Specs:
Etude
- 4x NVIDIA RTX3090 GPU
- 10-GbE connection to Local Cryo-EM Storage Server
- 2TB SSD, 32TB HDD storage
- 128 GB RAM, i9 10980XE 3.0GHz 18-core CPU
Nocturne
- 4x NVIDIA RTX3090 GPU
- 10-GbE connection to Local Cryo-EM Storage Server
- 2x 2TB SSD, 384 GB RAM
- 2x Intel Xeon Scalable Gold 5220R Processor 24-core GPU