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Advanced Light Microscopy and Spectroscopy Laboratory



Leica Confocal SP5 MP AFM

Main Use: Stimulated emission depletion pulsed scanning superresolution microscopy, confocal scanning microscopy of large and thick specimens

Set up for stimulated emission depletion (STED) microscopy for fluorescence scanning imaging below the diffraction limit of light resolution in two different color channels.

STED scanning uses two beams, one generated by stimulated light emission to deplete the fluorescence of certain fluorochromes for a fraction of a second. stimulated emission depletion (STED) beam at 750 nm, generated by attenuating a pulsed infrared beam from a MaiTai laser through a 100 meter fiber optic. This beam can be shaped in the form of a doughnut whose hole can be made smaller than the 200-300 nm limit of light resolution. A second beam from a either a 561 or 635 nm pulsed laser for excitation is aligned with the latter beam to pass through the hole and both beams are then be scanned across the specimen giving a much smaller effective excitation beam allowing resolution of details between 60-90 nm.

Equipment Highlight

  • Resonant scanner capable of 8000 Hz scanning speed (16000 Hz with bidirectional mode).
  • single photon lasers:
    a) Argon with 458, 476, 488, 496 and 514 nm excitation lines.
    b) 561 nm DPSS (yellow-green).
    c) 594 and 633 nm helium neon lasers.
  • DIC, 6 PMTs, 2 APDs, 2 NDDs.
  • CCD camera (for viewing IR).
  • MaiTai laser can also be run as a two photon laser for multiphoton scanning.
  • Software: Leica Application Suite–Advanced Fluorescence (LAS AF
  • For more product information, please visit Leica Microsystems