Equipment
Janelia Krios 1
This microscope was installed in 2011 and has undergone multiple rounds of hardware and software upgrades to keep pace with advances in the cryo-EM field. It is one of the few Krios systems equipped with a spherical aberration (Cs) corrector, reducing aberration from 2.7 mm to ~0.01 mm.
Originally configured with a film camera (still installed), it also included a pre-GIF Falcon I and a post-GIF Gatan US4000 CCD. As detector technology evolved, the US4000 CCD was replaced with a Gatan K2 Summit, and the Falcon I was upgraded to a Falcon II. In September 2019, the post-GIF K2 was replaced with a Gatan K3 camera. In December 2022, the Gatan Quantum energy filter was upgraded to a Gatan BioContinuum HD. The system now runs on Windows 10 with regularly updated data acquisition software.
Main Configuration
- High-brightness field emission gun (X-FEG)
- Spherical aberration corrector (Cs corrector)
- Pre-filter Ceta camera
- Energy filter: Gatan BioContinuum HD (GIF)
- Post-GIF Gatan K3 camera
- Scanning Transmission Electron Microscopy (STEM) capability
Main Applications
- Single-particle cryo-EM
- Cryo-electron tomography
Janelia Krios 2
Installed in 2013, this microscope has also been extensively upgraded. It was originally equipped with a standard FEG (s-FEG), later upgraded to an X-FEG. Initially configured for single-particle analysis and electron diffraction, it did not include an energy filter and used a Gatan K2 Summit detector and a TVIPS F416 camera.
In 2019, the TVIPS camera was replaced with a Gatan BioQuantum energy filter and a Gatan K3 detector. The system now runs on Windows 10 with regularly updated data acquisition software.
Krios2 will be retired in May 2026. A new Krios 5 will be set up in the same room. Stay tuned.
Main Configuration
- High-brightness field emission gun (X-FEG)
- Energy filter: Gatan BioQuantum (GIF)
- Pre-filter Gatan OneView camera
- Post-GIF Gatan K3 camera
Main Applications
- Single-particle cryo-EM
- Cryo-electron tomography
Janelia Krios 3
This microscope was installed during the COVID-19 pandemic and became operational in September 2021.
Main Configuration
- Cold field emission gun (C-FEG)
- Energy filter: Thermo Scientific Selectris X
- Pre-filter Ceta camera
- Post-filter Thermo Scientific Falcon 4i camera
- Scanning Transmission Electron Microscopy (STEM) capability
Main Applications
- Single-particle cryo-EM
- Cryo-electron tomography
Janelia Krios 4
Installed in 2025.
Main Configuration
- Cold field emission gun (C-FEG)
- Energy filter: Thermo Scientific Selectris X
- Pre-filter Ceta camera
- Post-filter Thermo Scientific Falcon 4i camera
Main Applications
- Single-particle cryo-EM
- Cryo-electron tomography
FEI Aquilos 2 Cryo FIB-SEM
Main Application
- Sample preparation for cryo-electron tomography
Capabilities
- Gallium ion beam milling of frozen hydrated samples into 100–300 nm lamella
- Cryo lift-out for thick samples
- Integrated fluorescence light microscopy (iFLM) for locating regions of interest
FEI Hydra Cryo Plasma FIB-SEM (cryo pFIB-SEM)
Main Applications
- Sample preparation for cryo-electron tomography
- Small-volume cryo-SEM imaging
Capabilities
- Plasma ion beam milling (Xe, Ar, O₂, N₂)
- Lamella preparation (100–300 nm) of frozen hydrated samples
- Cryo lift-out capability
- Integrated fluorescence light microscopy (iFLM)
- Cryo-SEM imaging
Leica Cryo CLEM Light Microscope
A wide-field fluorescence microscope for imaging cryo grids.
Main Application
- Identification of regions of interest for downstream cryo-FIB milling
Leica Cryo Confocal Microscope
A Leica Stellaris 8 confocal microscope with cryo capability.
Applications
- Identification of regions of interest in frozen hydrated samples
- Confocal imaging under cryogenic conditions
- Fluorescence lifetime imaging (FLIM)
SPT Labtech Chameleon System
A next-generation cryo-EM grid preparation system that dispenses nanoliter volumes onto self-wicking grids during plunging.
Plunge Freezers and Screening TEMs
The facility includes multiple plunge freezers and supporting equipment for room temperature TEM, cryo grid preparation and screening.
Available Instruments
- 2 FEI Mark IV Vitrobot plunge freezers
- Leica EM GP cryo plunger
- Leica EM GP2 cryo plunger
- Talos L120C TEM
- Tecnai F20 TEM
Services
The CryoEM Facility at Janelia Research Campus provides services to all HHMI labs and their collaborators. The facility is an HHMI-wide shared facility with state-of-the-art instrumentation and strong staff expertise, offering services to cover the entire CryoEM workflow. It hosts three FEI Titan Krios cryo transmission electron microscopes (cryo TEMs), an FEI Aquilos 2 cryo Focused Ion Beam Scanning Electron Microscope (cryo FIBSEM), multiple cryo grids preparation equipment and screening TEM microscopes.
Main services
- High-quality high-throughput single particle or cryo electron tomography (cryo ET) data collection on ready-to-image cryo grids prepared and provided by users
In this case, the facility does not help calibrate samples, prepare grids, milling thick samples or process data. Users need to prepare and screen cryo grids at their local facility before shipping cryo grids to Janelia. The purpose of this requirement is to ensure that the cryo grids and samples are of acceptable quality, to use microscope capacity at the Janelia cryoEM facility most responsibly. The user can either come to Janelia or work remotely. In either case, facility staff will operate the microscope, optimize imaging conditions/parameters, and set up automated data collection while users can focus on selecting targets for data collection. Both single particle and cryoET data collection are fully supported. - Cryo FIBSEM on thick frozen hydrate samples
For user-provided frozen hydrate samples that are too thick to image directly on a Krios cryo TEM such as cells frozen on an EM grid, we offer an FEI Aquilos2 Cryo FIBSEM and a Hydra plasma cryo FIBSEM to mill regions of interest into thin lamella. The lamella can then be loaded into a Krios cryoTEM microscope for imaging or tomography data collection. To help locate regions of interest before cryoFIB milling, a Leica Thunder cryoCLEM light microscope is available to image the cryo-EM grid. In addition to cryo FIB milling, the Hydra plasma FIBSEM can also be used to carry out small volume cryo SEM imaging on suitable samples.
- Collaboration work on other aspects of the cryoEM workflow
If needed, the facility can collaborate with an HHMI lab on other aspects of the cryoEM workflow such as cryo grids preparation and screening, image processing and 3D reconstruction. The lab can provide the facility a sample which is not ready for cryoEM imaging such as protein complexes in solution, cultured cells or tissues. The facility calibrates the sample and carries out portions or all of the cryoEM-related work on suitable samples, such as cryo grid freezing/screening, cryo FIB milling, data collection, image processing and 3D reconstruction.
Access
All HHMI labs and their collaborators are eligible to use the facility.
Getting Started
- New users should contact the facility to initiate registration
- Online scheduling system:
https://cryoem-scheduler.janelia.org/
For Registered Users
- Each month, the Janelia CryoEM Facility notifies all registered users via email when new booking time slots become available.
- Reserve instrument time through the online scheduler.
- Follow the provided guidelines for grid submission and instrument usage.
Contact
- Dr. Zhiheng Yu (Facility Director): yuz10@janelia.hhmi.org
- Dr. Rui Yan (CryoEM Aquisition Lead): yanr@janelia.hhmi.org
Quick Links
Our services are for HHMI labs and their collaborators.
Review policies and instructions before reserving time on equipment or sending any shipments.
Reserve time on our equipment.
Complete the Grid Submission Form before shipping cryo-EM grids for data collection.
Instructions for shipping cryo-EM grids.
Instructions for shipping solution samples.