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
Virus Imaging Core
Director: Tom Hope, PhD
The Virus Imaging Core provides state-of-the-art fluorescence imaging of HIV and HIV-infected cells and tissues. Four different imaging systems are available and are complemented by a toolbox of fluorescent and validated systems that allow us to image virus, viral proteins, cellular proteins, and infected cells during all stages of the HIV lifecycle. These capabilities are guided by more than a decade of experience in the development and utilization of cell biology approaches to illuminate HIV biology.
Instrumentation and Capabilities
Deconvolution Microscopy with DeltaVision
The Virus Imaging Core has three dedicated DeltaVision deconvolution microscopes. Each of the systems has different features that facilitate fluorescent imaging of live and fixed cells. System #1 has an environmental chamber to maintain viability and an EM-CCD camera for live cell imaging. It has two shutter controlled Xenon bulb light sources allowing near simultaneous imaging. A Coolsnap camera allows high resolution deconvolution imaging in fixed samples. It is also retrofitted with a spectral imaging camera to allow analysis of fluorescent markers in autofluorescence-rich samples such as tissues sections. System #2 also has an environmental chamber, EM-CCD, and Coolsnap camera with a solid-state illumination source to provide additional fluorophore utilization and near simultaneous two-color imaging. This system also contains an automated dichroic turret, 10 position emission filter wheel, and 7 position eyepiece filter wheel, which will allow us to take advantage of new fluor systems. System #3 has mercury bulb illumination and a Coolsnap camera allowing high-resolution deconvolution imaging. All of the microscopes are housed in biocontained rooms. Together these systems enable a diverse array of imaging protocols used in the study of HIV biology. To enhance our capabilities even further, we are upgrading two of our DeltaVision microscope systems. The upgrade will include: 1) Automated dichroic turret, 10 position emission filter wheel, and 7 position eyepiece filter wheel on System #1, and 2) an environmental chamber on System #3, which will give us live cell imaging capabilities on our one imaging system that lacks this capability.
Super-resolution Structured Illumination Microscopy with DeltaVision OMX
The virus imaging core has a dedicated DeltaVision OMX structured illumination microscope capable of super-resolution imaging. The system is equipped with multiple lasers capable of simultaneously imaging up to four wavelengths, allowing high speed fixed cell, live cell, and FRET imaging at resolutions of 90 nm in XY and 220 nm in Z. This imaging system will be housed within the Hope Lab in the Lurie Research Center.
The Virus Imaging Core has a dedicated Prairie 2-Photon microscope with a high intensity tunable infrared laser. The system is located within a dedicated room in a BSL-3 facility outfitted with an anesthesia machine and warming plate to facilitate intravital imaging of HIV infected cells in humanized mice.
145 Tbyte Server
The Virus Imaging Core has access to the Hope Laboratory 145 Tbyte CORAID server. This server provides secure mirrored storage of image files. The Virus Imaging Core also has access to image analysis software, including API SoftWoRx, Imaris Bitplane, Metamorph, and custom software developed in the IDL format.
Cell Imaging Facility
The Northwestern University Cell Imaging Facility (http://www.feinberg.northwestern.edu/cif/), located across the street from the Hope Laboratory/Virus Imaging Core, provides access to a diverse collection of imaging instruments on a reimbursement basis. Instruments available include: 2 Zeiss LSM 510 META laser scanning confocals, Zeiss PALM Laser Catapulting Microdissection System, Nikon C1Si spectral scanning confocal, Yokogawa spinning disk confocal microscope, TissueGnostics Tissue/Cell High Throughput Imaging and Analysis system, Olympus DSU diskscan system, JEOL 1220 transmission electron microscope, FEI Tecnai Sprit G2 120 kV TEM with 2-D tomography, Olympus OV-100 Whole-mouse imager, and an Olympus IV-100 intravital imaging system.
Fluorescence Imaging in Fixed Cells, Live Cells, and Living Animals.
As demonstrated in our publications, the Virus Imaging Core has extensive experience with fluorescent deconvolution imaging of fixed and live cells. Detailed methods and description of tools and protocols are described in the following publications generated in the current funding cycle, including: 1) Two-color live cell imaging is featured in Campbell et al., J. Cell Biol. (2007) 180 (3):549-561, 2) Single HIV virion imaging in fixed cells described in Hulme et al., Proc. Natl. Acad. Sci. (2011) 108(24):9975-9980. The core derives additional expertise in intravital imaging from interactions with members of the NIH funded Northwestern Interdepartmental Two-Photon Imaging Center (5P30NS054850-02).
Identifying Assembled Gag Complexes. Monoclonal antibody AG3 cannot recognize assembled or immature particles owing to epitope masking. Top: HeLa cell transfected with Gag-cherry and stained with AG3. Inset on right shows no green staining of VLPs. Bottom: Virus prepared with protease inhibitor cannot be stained with AG3 antibody. Images were taken in the Hope lab.