The mission of the Laboratory of Immunogenetics (LIG) Imaging Facility is to provide the necessary instrumentation, training, and technical support to allow principal investigators, postdoctoral fellows, and students to acquire and analyze high-resolution images of living cells at the level of individual molecules. Under the leadership of Dr. Joseph Brzostowski, the facility provides training to fellows and students and encourages communication among users to foment academic growth and solve common problems. It is the philosophy of the facility to maintain fluidity in the open design of microscopes to meet the changing needs of the investigators using the systems.
Instruments in the LIG Imaging Facility are available by appointment. Contact Dr. Brzostowski.
The facility supports a variety of light microscopy techniques to image live cells that include laser scanning confocal microscopy, spectral imaging, spinning disk confocal microscopy, total internal reflection microscopy, single particle imaging, and fluorescence lifetime imaging. The facility has recently acquired a 2-photon imaging system to visualize cells in live animals. While interested in the exotic, the facility also welcomes routine imaging of fixed samples. The facility supports six centralized computer workstations and software for image analysis.
Total internal reflection fluorescence microscopy (TIRFM) is a spatially limited imaging technique that is used primarily to visualize fluorescent molecules at or near the plasma membrane. Because the technique greatly minimizes out-of-focus, fluorescence and uses a fast, highly sensitive charge-coupled device (CCD) camera to detect low-level signal, it is possible to visualize and track the movement of single-molecule fluorophores. See Total Internal Reflection Fluorescence Microscope for an overview of the method.
We have successfully used TIRFM toward understanding a wide range of signal transduction events that regulate immune cell biology. To highlight the technique, examples are shown below from experiments performed in LIG with B cells, a target of natural killer cells, and Dictyostelium discoideum—an amoeboid cell that models the chemotactic movement of neutrophils and macrophages.
Presently, we are performing stochastic optical reconstruction microscopy (STORM) super-resolution imaging experiments using the TIRFM system to understand the topography of the B c-ell receptor and its co-receptors in the plasma membrane.
The facility has two TIRFM systems (TIRF-1 and TIRF-2) that offer both overlapping and unique functionality.
TIRF-1TIRF-1 is located in Twinbrook II, Room 223-C.
TIRF-2TIRF-2 is located in Twinbrook II, Room 223-B, and has the same capabilities as TIRF-1, as well as some additional features.
Spinning disk confocal microscopy is a "wide-field" technique (a CDD camera captures the entire field of view). In contrast to the relatively slow, point-scanning method of a conventional confocal microscope (capture rate of about one frame per second), the spinning disk can acquire images at video rate (30 frames per second) or greater. In conjunction with a piezo driven Z-axis stage, a single optical slice can be captured in about 50 milliseconds, allowing the user to acquire a 3D image of a typical cell in less than one second. An example of a 3D time-lapse acquisition of ligand-induced polymerization of fluorescently labeled F-actin in a live Dictyostelium discoideum cell is shown below.
Our Yokogawa CSU-X1 spinning disk confocal unit supplied by Solamere Technologies, is located in Twinbrook II, Room 223-C.
The CSU-X1 is a technologically advanced unit, having a computer-controlled dichroic mirror, emission filter wheel, and variable-speed disk motor that minimizes scan line artifacts due to mismatched exposure times.
The LIG Imaging Facility has two laser scanning confocal systems to image live cell specimens. Both systems are Zeiss inverted microscope platforms and can perform spectral imaging. The Zeiss LSM 510 Meta system is visible a light-only system and serves as the workhorse for the facility. The Zeiss LSM 780 is a multi-use, state-of-the-art system equipped with a highly sensitive spectral GaAsP and cooled PMTs, ideal for live cell imaging experiments. The LSM 780 is also equipped with a pulsed infrared laser and specialized detectors for fluorescence lifetime imaging (FLIM) and intravital imaging experiments.
Zeiss LSM 510 MetaThe Zeiss LSM 510 is located in Twinbrook II, Room 223-D.
Zeiss LSM 780The Zeiss LSM 780 is located in Twinbrook II, Room 223-F.
An integral part of the imaging facility is the computer workstation corral for data analysis, located in Twinbrook II, Room 201. There are five PC workstations and one Mac workstation. Users have access to a data storage server that is backed up daily. While the corral is a quiet, open office space for work and study, it also promotes conversation among users to discuss data and technical issues. The data analysis corral supports a wide range of image analysis software packages:
Imaging facility group members have written several book chapters that detail methods for acquiring and analyzing FRET and single particle data using TIRFM and FRET by confocal microscopy.
Davey A, Liu W, Sohn HW, Brzostowski JA, Pierce SK. Understanding the initiation of B cell signaling through live cell imaging. Methods Enzymol. 2012;506:265-90.
Sohn HW, Tolar P, Brzostowski J, Pierce SK. A method for analyzing protein-protein interactions in the plasma membrane of live B cells by fluorescence resonance energy transfer imaging as acquired by total internal reflection fluorescence microscopy. Methods Mol Biol. 2010;591:159-83.
Xu X, Brzostowski JA, Jin T. Monitoring dynamic GPCR signaling events using fluorescence microscopy, FRET imaging, and single-molecule imaging. Methods Mol Biol. 2009;571:371-83.
Tolar P, Meckel T. Imaging B-cell receptor signaling by single-molecule techniques. Methods Mol Biol. 2009;571:437-53.
Brzostowski JA, Meckel T, Hong J, Chen A, Jin T. Imaging protein-protein interactions by Förster resonance energy transfer (FRET) microscopy in live cells. Curr Protoc Protein Sci. 2009 Apr;Chapter 19:Unit19.5.
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Last Updated September 25, 2014