Cryo-EM Services

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.
 

ON THIS PAGE

Sample Preparation  |  Data Collection and Processing

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 the cryo-EM lead:

Melody Campbell, Ph.D.
Scientific Director, Cryo-EM

Sample Preparation Services

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

Cryo-EM 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 recommended buffer conditions and 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
Researcher training another researcher on a piece of scientific equipment
Dr. Betty Shen sets up for a grid freezing session at the Vitrobot Mark IV station. Photo by Robert Hood / Fred Hutch News Service

Services

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Thin Carbon Coating of Grids

We provide thin carbon coating of grids for negative-stain or cryo-EM studies. The standard turnaround time to coat negative-stain or cryo-EM grids with a thin layer of carbon is five business days. 

Negative Staining

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.

Researcher negative staining
We offer all supplies for negative-staining grids except grids and samples. Photo by Robert Hood / Fred Hutch News Service
Cryo-EM Grid Freezing

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 grids, samples and buffers.

Clipping and Loading Autogrids

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.

Researcher working with equipment
Dr. Caleigh Azumaya clips and loads autogrids for screening and data collection in the Glacios microscope. Photo by Robert Hood / Fred Hutch News Service
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Equipment

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Negative-Stain Bench

The negative-stain bench provides users with everything needed to make and stain their own sample for electron microscopy. This includes core-provided tweezers, uranyl formate stain, continuous carbon grids and grid boxes for storage, as well as use of the glow discharger and carbon coater.

Related service: Negative staining

Denton BenchTop Turbo Coating System and Glow Discharger

This thin-film deposition system provides full-range, low-voltage thermal carbon evaporation and can produce fine-grained thin films via sputtering. The instrument allows transmission electron microscopy grids to be cleaned in plasma using the AC glow discharge option.

Related service: Thin carbon coating of grids

Scientific piece of equipment
Photo by Robert Hood / Fred Hutch News Service
Thermo Fisher Scientific Mark IV Vitrobot

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

Scientific piece of equipment
Photo by Robert Hood / Fred Hutch News Service

Data Collection and Processing Services

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 the high-performance computing cluster maintained by Fred Hutch's Center IT.

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

Training in Data Collection and Processing

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.

Researchers working on a computer
Dr. Caleigh Azumaya explains to Dr. Nick Hurlburt what to look for on the Glacios microscope. Photo by Robert Hood / Fred Hutch News Service

Services

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Negative-Stain Data Collection

Our staff will collect a full dataset on prepared negative-stain grids using data-collection parameters that we have decided and agreed upon during consultation with users. We will provide micrographs to users immediately following collection.

Researcher working with equipment
Dr. Melody Campbell (foreground) inserts a negative-stain sample while Dr. Caleigh Azumaya prepares to set up data collection. Photo by Robert Hood / Fred Hutch News Service
Two-Dimensional Averages

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.

Microscopic image
Two-dimensional class averages of integrin and latent TGFβ. Image courtesy of Dr. Melody Campbell
Image-Processing Office Hours

Users can stop in via video chat to ask any processing questions from 3-4 p.m. every Tuesday and Thursday.

Please access image-processing office hours through Zoom.

Equipment

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Talos L120C Transmission Electron Microscope and Ceta-M CMOS 16M Camera

This transmission electron microscope, or TEM, from Thermo Fisher Scientific is operated at an accelerating voltage of 120 kV and is equipped with a 4k x 4k CMOS detector that can record data at up to 40 frames per second. The microscope is fitted with a long-duration dewar that allows for up to four days of uninterrupted microscope usage, enabling fully automated overnight data collection. This microscope and detector support negative-stain and cryo-EM screening and data collection.

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
  • 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.

Scientific piece of equipment
Image courtesy of Thermo Scientific
200 kV Glacios Transmission Electron Microscope and Gatan K3 Direct Electron Detector

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
Scientific piece of equipment
Image courtesy of Thermo Scientific
Mercury GPU208 2U Server

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, particle picking and 2D classification.

Server specs:

  • Dual Xeon Gold 6254 3.1 GHz 18-core CPU
  • 4x PNY NVIDIA RTX A6000 GPU
  • 384 GB RAM
Mercury RM436 4U Rackmount Server

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

Questions about our cryo-EM services or how to schedule with us?