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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 Dr. Brzostowski.

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, 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.

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 211.

Features

  • Computer-controlled Olympus IX 81 inverted microscope
  • 60X, 100X, and 150X TIRFM lenses
  • MetaMorph software operating system
  • 512 x 512 electron-multiplying CCD, (Cascade IIB, 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 201G, and has the same capabilities as TIRF-1 as well as some additional features.

Features

  • 405 nm laser to perform photoactivation, photoconversion experiments, and photoactivated localization microscopy (PALM) experiments
  • Pulsed, dye-cell laser (Micropoint, Photonic Instruments) to perform targeted, subcellular photoactivation, photoconversion, and fluorescence recovery after photobleaching (FRAP) experiments in the TIRFM imaging plane
    • Three wavelengths are available: 404 nm, 435 nm, and 551 nm
  • 1024 x 1024 electron-multiplying CCD (Cascade IIB, Photometrics)
  • Six laser excitation lines (405, 440, 488, 514, 561, and 640 nm)

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. 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 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). 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.

Features

  • Computer-controlled Olympus IX 81 inverted microscope
  • 60X, 100X, and 150X lenses
  • MetaMorph software operating system
  • 512 x 512 electron-multiplying CCD (QuantEM, 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
    • 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
    • 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 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 Meta
The Zeiss LSM 510 is located in Twinbrook II, Room 201G.

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 (PMT) detector, two standard PMT detectors, and one 8-channel spectral detector
    • Spectral detector 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, CO2, and humidity
    • Supports mammalian cells

Zeiss LSM 710
The Zeiss LSM 710 is located in Twinbrook II, Room 225.

Features

  • Multi-color imaging from near ultraviolet (DAPI-like dyes) through the visible spectrum
  • Coherent “Chameleon” Ultra II Ti Sapphire multi-photon 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, two standard PMT detectors, and one 32-channel spectral detector
    • 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
    • Supports mammalian cells
  • Two high-speed hybrid GaAsP detectors (Becker and Hickl, HPM-100-40) for FLIM and time-correlated single photon counting (TCSPC)
  • Two-channel non-descanned detection (NDD) units for deep-tissue imaging
    • Live animal imaging to be announced

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 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:

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

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Last Updated November 09, 2012