Joseph Brzostowski, Ph.D.Twinbrook II, Room 20112441 Parklawn DriveBethesda, MD 20892-8180Phone: 301-443-2967Fax: firstname.lastname@example.org
The mission of the 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 Dr. Brzostowski’s leadership, 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, background 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 Microscopy Resource Center for an overview of the technique.
We have successfully used TIRFM toward understanding a wide range of signal transduction events that regulate immune cell biology. Shown below, to highlight the technique, are examples 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.
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 211.
TIRF-2TIRF-2 is located in Twinbrook II, room 201G, and has the same capabilities as TIRF-1 as well as some additional features.
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. In press.
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.
Xu X, Brzostowski JA, Jin T. Using quantitative fluorescence microscopy and FRET imaging to measure spatiotemporal signaling events in single living cells. Methods Mol Biol. 2006;346:281-96.
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). In conjunction with a piezo driven Z-axis stage, a Z slice can be captured in about 70 milliseconds, allowing the user to acquire a 3D image of a typical cell in less than one second.
The TIRF-1 microscope, located in Twinbrook II, room 213, is a dual-platform system that allows the user to image by TIRFM or confocal microscopy. Attached to the side port of the Olympus IX81 microscope is a Yokogawa CSU-X1 spinning disk confocal unit supplied by Solamere Technologies.
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 our standard confocal system and serves as the workhorse for the facility. The Zeiss LSM 710 is a multi-use, state-of-the-art system equipped with a multi-photon laser.
Zeiss LSM 510 MetaThe Zeiss LSM 510 is located in Twinbrook II, Room 201G.
Zeiss LSM 710The Zeiss LSM 710 is located in Twinbrook II, Room 225.
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 an unlimited 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:
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Last Updated November 09, 2012