Equipment/Service

Equipment

Janelia Krios 1

This microscope was set up in 2011. It has gone through multiple rounds of hardware and software upgrades to keep up with new developments in the cryoEM field. This Krios was one of the few in the world that is equipped with a Cs corrector to reduce spherical aberration of the objective lens from 2.7 mm to ~0.01mm. It came with a film camera, which is still on the scope. At the time of purchase direct detectors were still in early days of development and films were still in use. The other original cameras that came with the microscope were a pre-GIF Falcon I and a post-GIF Gatan US4000 CCD. Later the US4000 CCD was replaced with a Gatan K2 Summit camera and the Falcon I was replaced with a Falcon II camera. In September 2019, the post-GIF K2 camera was replaced with a Gatan K3 camera. In December 2022, the Gatan Quantum energy filter was replaced with a Gatan BioContinuum HD energy filter. The operating system was upgraded to Windows 10 and the main microscope operating software and data collection software have been frequently upgraded.

Main configuration

• High-brightness field emission gun (X-FEG)
• Spherical aberration corrector (Cs corrector)
• Energy filter: Gatan Image Filter (GIF) BioContinuum HD
• Post-GIF  Gatan K3 camera
• Volta Phase Plate

Main applications

• Single particle cryoEM
• Cryo electron tomography

Janelia Krios 2

This microscope was set up in 2013 and has been upgraded frequently. It came with a standard field emission gun (s-FEG) which was later upgraded to an X-FEG. This Krios was originally configured for single particle data collection and electron diffraction. There was no GIF and the main camera set up was a Gatan K2 Summit detector directly under the projection chamber followed by a TVIPS F416 camera. In  2019 the TVIPS camera was replaced with a GIF BioQuantum energy filter and Gatan K3 camera. The operating system was upgraded to Windows 10 and the main microscope operating software and data collection software have been frequently upgraded.

Main configuration

• High-brightness field emission gun (X-FEG)
• Energy filter: Gatan Image Filter (GIF) BioQuantum
• Pre-filter Gatan Oneview camera
• Post-GIF  Gatan K3 camera
• Volta Phase Plate

Main applications

• Single particle cryoEM
• Cryo electron tomography

Janelia Krios 3

This microscope was set up during the COVID pandemic and came online in September 2021.

Main configuration

• Cold field emission gun (C-FEG)
• Energy filter: Thermo Scientific Selectris X
• Pre-filter Ceta camera
• Post-energy filter Thermo Scientific Falcon 4i camera 
• Volta Phase Plate

Main applications

• Single particle cryoEM
• Cryo electron tomography

FEI Aquilos 2 Cryo FIBSEM

Main application

• Sample preparation platform for cryo electron tomography
• Use focused ion beam (Ga ion)  to mill regions of interest on frozen hydrate samples with a thickness up to 50 micrometers into 100-300 nm thick lamella
• cryo lift-out capability to work on thick frozen hydrate  samples
• integrated fluroscence light microscope (iFLM) to help find region of interest for milling

FEI Hydra cryo plasma FIBSEM (cryo pFIBSEM)

Main application

• Sample preparation platform for cryo electron tomography
• small volume cryo SEM imaging on suitable samples
• Use plasma ion beam generated from one of four gases (Xe, Ar, O₂ and N₂) to mill regions of interest on frozen hydrate samples with a thickness up to 50 micrometers into 100-300 nm thick lamella
• cryo lift-out capability to work on thick frozen hydrate  samples
• integrated fluroscence light microscope (iFLM) to help find region of interest for milling

Leica Cryo CLEM light microscope

Autogrids can be used directly on the system that can be later transferred to a cryo FIBSEM. 

Main application

• Identify regions of interest in cells frozen on a cryo-EM grid using wide field and/or fluorescence light microscopy that may be used later to aid cryoFIB milling.

STP Labtech Chameleon System

This is a new generation cryo-EM grids preparation equipment. It dispenses a small volume (a few nanoliters) of sample onto a self-wicking grid while the grid is being plunged. For certain samples it may help reduce negative effects at the air-water interface.

Plunge freezers and screening TEMs

The facility also hosts multiple plunge freezers and other small equipment for making cryoEM grids. A Tecnai Spirit Biotwin TEM and a Tecnai F20 TEM  can be used to image negative stain grids or to screen cryo grids.

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

1. 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. 
2. 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.

3. 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 have access to the Janelia CryoEM facility. For new users, they need to first contact the facility to initiate the process of registering. The facility provides an online scheduler for users to register. https://cryoem-scheduler.janelia.org/

If a registered user has ready-to-image cryo grids, they can follow the guideline on the scheduler and book equipment time themselves. If a user needs more help, has technical questions or wants to initiate a collaboration with the cryoEM facility, they can either contact the facility director, Dr. Zhiheng Yu, at yuz10@janelia.hhmi.org or email cryoemfacility@janelia.hhmi.org.