Twinbrook Imaging Facility Systems

TIRF Laser

TIRF Laser

Credit: NIAID
The facility supports a number of light microscopy techniques to image live cells that include laser scanning confocal microscopy, spectral imaging, spinning disk confocal microscopy, FRET, total internal reflection fluorescence microscopy, single molecule tracking, super-resolution STORM imaging, Airyscan super-resolution imaging, fluorescence lifetime imaging, fluorescence correlation spectroscopy, and intravital imaging. While we may have a penchant for the exotic, the facility whole-heartedly welcomes routine imaging of fixed samples and supplies a host of nail polish hues to adorn users' slides.

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.

The facility has two TIRFM systems for live or fixed samples: a home-built rig using an Olympus IX 81 base, and a recently acquired (2017) Nikon N-STORM 4. Both systems can perform super-resolution STORM and PALM imaging experiments.

Olympus TIRF

Located in Fishers 5625, room 4S06E.

Features

  • Computer-controlled Olympus IX 81 inverted microscope
  • 60X, 100X, and 150X TIRFM lenses
  • MetaMorph software operating system
  • Liquid cooled 512 x 512 electron-multiplying CCD, (Evolve Delta, Photometrics)
    • Detects single fluorophores
    • Acquires images at up to 60 frames per second
  • Six laser excitation lines (405, 440, 488, 514, 561, and 640 nm)
  • Computer-controlled TIRF angle
  • 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
  • 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

Nikon N-STORM 4

Located in Fishers 5625, room 4S06E.  This system is state-of-the-art and can perform up to three color, three dimensional, super-resolution STORM or PALM imaging.

Features

  • Computer-controlled Nikon Eclipse Ti2
  • 100XTIRFM lens
  • Nikon Elements software operating system
  • Two 512 x 512 electron-multiplying CCD cameras, (iXon Ultra, Andor and Evolve Delta, Photometrics)
    • Detects single fluorophores
    • Acquires images at up to 60 frames per second
  • Four laser excitation lines (405, 488, 561, and 640 nm)
  • Computer-controlled TIRF angle
  • Differential interference contrast (DIC)/Nomarski imaging
  • LED Epi-illumination
  • 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
  • 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

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 laser-scanning 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 fast, 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 mammalian cell in less than one second.

Located in Fishers 5625, room 4S06B, the facility has a Yokogawa CSU-X1 attached to a motorized Olympus IX 81. 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. Presently, we have the MicAO, a newly developed adaptive optics device that can improve the emission light path by >100% by correcting aberrations (for details go to – http://www.imagine-optic.com/).

Features

  • Computer-controlled Olympus IX 81 inverted microscope
  • 100X 1.35 NA Silicone Oil
  • 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)
  • DIC/Nomarski imaging
  • Environmental control: heat, CO2, and humidity
  • 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
  • MicAO adaptive optics device

Laser Scanning Confocal Systems

The Twinbrook Imaging Facility has two state-of-the-art laser scanning confocal systems to image live cell specimens. The Zeiss LSM 780 is a multiplatform system capable of spectral imaging, intravital imaging, fluorescence lifetime measurements and fluorescence correlation spectroscopy. The LSM 880 is equipped with the super-resolution Airyscan detector and the FAST system, allowing it to achieve video rate recording speeds with a single color.

Zeiss LSM 880 Airyscan with FAST

Located in Fishers, room 4S06A.

Features

  • Multi-color imaging from near ultraviolet (DAPI-like dyes) through the visible spectrum
  • Five excitation laser lines (405, 488, 514, 561, and 633 nm)
  • One transmitted light photomultiplier (PMT) detector, one standard PMT, one GAsP PMT, and one cooled PMT for increased sensitivity for red fluorophores
  • 32 channel Airyscan detector for ~2x resolution improvement (resolves structures at ~125nm)
  • FAST system to increase scanning speed 4x, maintaining a 1.5x resolution improvement and providing up to 27 frames per second acquisition time for a single color. 
  • 20, 40, 63, and 100x objective lenses
  • Computer-controlled micromanipulator and microinjector for chemotaxis experiments
  • OKOlabs environmental control: heat, CO2, and humidity

Zeiss LSM 780 with Coherent Chameleon Ultra II

The Zeiss LSM 780 is located in Fishers 5625, room 4S06F.

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, one standard PMT, one 32-channel GAsP spectral detector and one cooled PMT for increased sensitivity for red fluorophores
    • 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
  • Environmental control: heat, CO2, and humidity
  • 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. LSM Inverter arm and 20x water dipping or 40x water (coverslip) lenses for intravital imaging
  • Fluorescence correlation spectroscopy
Content last reviewed on October 3, 2017