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Joseph Brzostowski, Ph.D.
Twinbrook II, Room 201
12441 Parklawn Drive
Bethesda, MD 20892-8180
Phone: 301-443-2967
Fax: 301-402-0259
brzostowskij@niaid.nih.gov


Laboratory of Immunogenetics

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LIG Imaging Facility

Photo of lab

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 Joseph Brzostowski, Ph.D. 

Imaging and Data Analysis Systems

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.

 

Olympus IX81 TIRFM Systems

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.

Single BCR molecules labeled with anti-IgM-Cy3 Fab on bilayers containing NIP14.
Tolar P, Hanna J, Krueger PD, Pierce SK. The constant region of the membrane immunoglobulin mediates B cell-receptor clustering and signaling in response to membrane antigens. Immunity. 2009 Jan 16;30(1):44-55.
Disruption of actin filaments in 721.221 cells increases the mobility of ICAM-1.
Gross CC, Brzostowski JA, Liu D, Long EO. Tethering of intercellular adhesion molecule on target cells is required for LFA-1-dependent NK cell adhesion and granule polarization. J Immunol. 2010 Sep 1;185(5):2918-26.
Imaging of Gβ-YFPs on the membrane of a live cell at 30 frames per second.
Xu X, Meckel T, Brzostowski JA, Yan J, Meier-Schellersheim M, Jin T. Coupling mechanism of a GPCR and a heterotrimeric G protein during chemoattractant gradient sensing in Dictyostelium. Sci Signal. 2010 Sep 28;3(141):ra71.

The facility has two TIRFM systems (TIRF-1 and TIRF-2) that offer both overlapping and unique functionality.

TIRF-1
TIRF-1 is located in Twinbrook II, Room 223-C.

Features
  • Computer-controlled Olympus IX 81 inverted microscope
  • 60X, 100X, and 150X TIRFM lenses
  • MetaMorph software operating system
  • 512 x 512 electron-multiplying CCD, (Evolve Delta, Photometrics)
    • Detects fluorophores at the single molecule level
    • Acquires images at video rate (30 frames per second) or faster
  • Five laser excitation lines (440, 488, 514, 568, and 647 nm)
    • 440 laser can be used for cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) Förster resonance energy transfer experiments (FRET)
  • Computer-controlled TIRFM angle
    • Optimizes the TIRFM imaging plane for multicolor experiments
  • Optical splitter, DualView (Photometrics)
    • Permits the simultaneous acquisition of two colors
  • Differential interference contrast (DIC)/Nomarski imaging
  • Interference reflection microscopy (IRM)
    • A specialized technique to visualize a cell’s contact area on the coverslip for correlation to the TIRFM imaging plane
  • Environmental control: heat, CO2, and humidity
    • Supports mammalian cells and multi-day experiments
  • Auto-focus laser
    • Permits long time-lapse imaging
  • Automated X, Y, Z stage
    • Permits the capture of multiple images in a chamber over a time-lapse by returning precisely to the same spot

TIRF-2
TIRF-2 is located in Twinbrook II, Room 223-B, and has the same capabilities as TIRF-1, as well as some additional features.

Features
  • STORM imaging experiments performed using two excitation wavelengths at 488 and 640 nanometers
  • 405 nm laser to perform photoactivation, photoconversion, and photoactivated localization microscopy (PALM) experiments
  • 512 x 512 electron-multiplying CCD (Evolve Delta, Photometrics)
  • Six laser excitation lines (405, 440, 488, 514, 561, and 640 nm)

Spinning Disk Confocal Microscope

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.

Features
  • Computer-controlled Olympus IX 81 inverted microscope
  • 60X, 100X, and 150X lenses
  • MetaMorph software operating system
  • 512 x 512 electron-multiplying CCD (Evolve Delta, Photometrics)
    • Acquires images at video rate (30 frames per second)
  • Five laser excitation lines (440, 488, 514, 568, and 647 nm)
    • 440 laser can be used for CFP/YFP FRET
  • Optical splitter, DualView (Photometrics)
    • Permits the simultaneous acquisition of two colors
  • DIC/Nomarski imaging
  • Environmental control: heat, CO2, and humidity to support mammalian cells and multi-day experiments.
  • Auto-focus laser
    • Permits long time-lapse imaging
  • Automated X, Y, Z stage
    • Permits the capture of multiple images in a chamber over a time-lapse by returning precisely to the same spot
  • Z axis driven by a piezo motor for extremely fast 3D imaging

Laser Scanning Confocal Systems

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 Meta
The Zeiss LSM 510 is located in Twinbrook II, Room 223-D.

Features
  • Multi-color imaging in the visible spectrum only (cannot perform DAPI imaging)
  • Six excitation laser lines (458, 488, 514, 543, and 633 nm)
  • One transmitted light photomultiplier tube (PMT) detector, two standard PMT detectors, and one 8-channel spectral detector
    • that can be used as a standard PMT for multi-color imaging
  • Simultaneous spectral imaging of 8 channels (32 channels can be obtained with four scans)
  • 10, 20, 40, 63, and 100x objective lenses
  • Computer-controlled micromanipulator and microinjector for chemotaxis experiments (can also be used for microinjection)
  • Environmental control: heat, and humidity
    • to support mammalian cells

Zeiss LSM 780
The Zeiss LSM 780 is located in Twinbrook II, Room 223-F.

Features
  • Multi-color imaging from near ultraviolet (DAPI-like dyes) through the visible spectrum
  • Coherent “Chameleon” Ultra II Ti Sapphire infrared pulsed laser
    • Tunable from 680 to 1080 nm
    • Supports fluorescence lifetime imaging (FLIM) and deep-tissue imaging
    • Can be used for standard confocal imaging
  • Six continuous wave excitation laser lines (405, 440, 488, 514, 561, and 633 nm) for standard confocal imaging
    • 405 nm line supports photoactivation experiments
  • One transmitted light PMT detector, one standard PMT detector, one 32-channel GaAsP spectral detector, one cooled PMT detector
    • The spectral detector can be used as a standard PMT for multi-color imaging
  • Simultaneous spectral imaging of 34 channels
  • 10, 20, 40, 63, and 100x objective lenses
  • Computer-controlled micromanipulator and microinjector for chemotaxis experiments (can also be used for microinjection)
  • Environmental control: heat, CO2, and humidity to support mammalian cells
  • Two high-speed hybrid GaAsP detectors (Becker and Hickl, HPM-100-40) for FLIM and time-correlated single photon counting (TCSPC)
  • One standard and one binary GaAsP (BiG) non-descanned detection (NDD) units for four channel deep-tissue/intraviral imaging experiments

Workstations and Analysis Software

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:

  • Dynamic Information Architecture System (DIAS) (cell tracing/tracking)
  • ImageJ
  • ImagePro Analyzer
  • ImagePro Plus 3D
  • Imaris (supported through NIAID core imaging facility)
  • Matlab
  • MetaMorph
  • Zeiss Axiovision
  • Zeiss ZEN

Selected Publications

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