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
Tissue EM Core
Director: Pamela Bjorkman
Manager: Mark Ladinsky
The Tissue EM Core performs high-quality preservation of HIV-infected tissues from both humanized mouse model systems and actual human tissues from biopsies and explants. These samples are plastic-embedded, serially sectioned and imaged for tomographic analysis in either of two electron microscopes. Tomographic reconstructions provide three-dimensional, high-resolution studies of wild-type HIV within actual infected tissues and organs and may shed light on the fine structures involved in HIV budding and evasion from HAART therapy. The Core has most equipment necessary for processing, imaging and analyzing tissue samples. The core has effectively unlimited access to two tomography-ready electron microscopes: A FEI Tecnai T12 (120KeV) microscope for sections ≤200nm in the Caltech Broad Center EM Suite (see EM Tomography Core, Grant Jensen Director) and a newly retrofitted Tecnai TF30-FEG (300KeV) microscope for 300-400nm sections located in the Caltech Material Sciences Department.
Instrumentation and Capabilities
The core is equipped with its own Bal-Tec HPF-010 High-Pressure Freezer for cryo-immobilization of tissue samples and an older Leica AFS Freeze-Substitution Machine for low-temperature tissue processing. It also has a Leica UC6/FC6 ultramicrotome for sectioning of both plastic-embedded and vitreously frozen samples, and access to two other ultramicrotomes for plastic sectioning in the Jensen EM Suite. Other hardware needed for sample preparation, including phase-contrast and dissecting light microscopes, and carbon evaporation and glow-discharging machines, are available within the Core. During the current funding cycle, we will obtain a Leica EM AFS2 freeze substitution instrument, which will greatly enhance our sample throughput and provide better temperature control and biohazard containment. We will also purchase a Leica CED030 carbon thread evaporator, which will increase our ability to reproducibly deposit thin carbon films on EM grids, and thereby improve image quality.
The two tomography-ready electron microscopes described above are equipped with 2k x 2k CCD cameras, fast multiport readout systems and the software necessary for high-throughput, automated acquisition of dual-axis tomographic datasets (SerialEM, UCSF-Tomography, FEI Tomo and Leginon). The Core Manager has expertise with both microscope systems and he will operate the instruments and train other Core members in their use. In addition, the Core owns a Fischione Model 2010 high-tilt/rotation specimen holder, which facilitates accurate sample rotation and even faster acquisition of dual-axis tomograms. With this hardware, datasets can be acquired in as little as 40 minutes.
Tomogram Image Processing
The Core will be using the IMOD software package for most tomogram processing and analysis. This software runs on most desktop computer platforms. The Core Manager has been working IMOD since its conception in 1994 and serves as a beta-tester for new versions. Large-format, montaged and serial-section tomographic reconstructions require computers with considerably more memory and storage space. The Core currently has several Macintosh computers capable of the task, including a 12-core MacPro with 64GB of memory and over 10TB of storage space.