Bacterial Protein Expression Core
Cell Biology and Imaging Core
EM Crystallography Core
EM Tomography Core
Eukaryotic Protein Expression Core
Fluorescence Spectroscopy Core
Protein Interactions Core
Protein NMR Spectroscopy Core
RNA Structure and Dynamics Core
Tissue EM Core
Virus Imaging Core
X-ray Crystallography Core
EM Tomography Core
Director: Grant Jensen, PhD
The EM Tomography Core produces 3D reconstructions of unique samples such as individual virions or cells to macromolecular (~2-6 nm) resolution. Two cryoelectron microscopes are used, a 300-kV Polara and a 120-kV T12, both from the FEI Company. Samples are either plunge-frozen in liquid ethane or high-pressure frozen and then cryosectioned. Tilt-series are acquired automatically and then combined computationally to produce the 3-D reconstructions.
Instrumentation and Capabilities
The Caltech cryoEM facility houses two TEMs: a 300kV, energy-filtered, helium-cooled, FEG G2 Polara and a 120kV Tecnai 12. The Polara has a field-emission gun, a Gatan GIF energy filter with a 4k x 4k "K2 Summit" direct detector, a 1k x 1k "pre-GIF" retractable CCD, off-axis "pre-GIF" and GIF TV cameras, and a helium-cooled CompuStage. The apertures, stage, electron optics, and all other controls are fully computer-controlled so that the entire instrument can be operated from a networked remote operation station. A new "flip-flop" rotation stage allows tilt-series to be recorded along two orthogonal axes without removing the sample from the column vacuum. The multispecimen holder transfers 6 grids at a time in a nearly contamination-free procedure.
The Tecnai 12 TEM has a LaB6 filament, a 2k x 2k Ultrascan CCD, a TV-camera, a computer-controlled CompuStage, and a high-tilt holder for tomography. It is fully equipped for cryo work with a Gatan cryoholder, a cryotransfer stage, and a dry pumping station. Uninterruptible power supplies are used for both microscopes.
Fluorescent Light Microscopes for Correlated fLM/EM Studies
A Nikon fluorescent light microscope (fLM) has been purchased for correlated fLM and EM studies. This microscope is equipped for bright field, phase contrast, differential interference contrast, epifluorescence, and time-lapse imaging. With a motorized stage, CCD, and Metamorph Premier software, it can autofocus, record multi-modal images in rapid sequence, capture montage overviews, and collect time lapse series of multiple targets simultaneously. Methods have been developed to attach cells to an EM grid, observe them by fLM, quickly plunge-freeze them on the EM grid, locate the same cells in the electron microscope, and record tomograms.
A second Nikon microscope with an EMCCD is equipped for “super-resolution” photoactivation localization microscopy (PALM). It is a high-end, fully-automated motorized inverted microscope, with an oil immersion, 100x TIRF objective, an extra-long-working-distance (ELWD), 60x air objective, and Nikon’s exclusive Perfect launch system, which houses three 100mW lasers with wavelengths at 405nm, 488nm and 561nm, and can provide either TIRF or wide field illumination. Comprehensive imaging software NIS-Elements offers integrated control of the microscope, illuminations and camera, and allows automated multi-modal imaging, such as bright field, phase contrast, epifluorescence, z-stack, time lapse, stage scan and TIRF. The microscope is also adapted to work with FEI’s correlative cryo-stage, which allows for observation of vitrified biological samples on standard EM grids.
Sample Preparation and Other Auxiliary EM Equipment
A Vitrobot III plunge-freezer and two portable, in-house, gravity-driven plunge-freezers are available for preparing frozen-hydrated samples. A Balzers high-pressure freezer, Leica UltraCUT microtome with cryosectioning attachment, Leica low-temperature embedding system, and Leica glass knife-maker are available for producing fixed sections.
Image Processing Pipeline
Tilt-series are recorded with either the Leginon software (developed in part at Caltech) or UCSF Tomo package. As tilt-series finish, they are automatically moved into the Jensen Lab tomography database (which now organizes approximately 20,000 tomograms) and processed with Raptor, a program for automatic 3-D reconstruction. Tilt-series and Raptor reconstructions are then visually inspected and sometimes re-processed by hand with IMOD. Subtomogram averaging and CTF correction are done with Bsoft or PEET. Segmentations and visualizations are done with 3dmod, Amira, Chimera. Animations are done with Maya, and custom operations are performed using various in-house software. Final tomograms and associated files are archived in the database.