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Resonant-scanning dual-color STED microscopy with ultrafast photon counting: A concise guide

Methods, ISSN: 1046-2023, Vol: 88, Page: 48-56
2015
  • 18
    Citations
  • 0
    Usage
  • 47
    Captures
  • 0
    Mentions
  • 7
    Social Media
Metric Options:   Counts1 Year3 Year

Metrics Details

  • Citations
    18
  • Captures
    47
  • Social Media
    7
    • Shares, Likes & Comments
      7
      • Facebook
        7

Article Description

STED (stimulated emission depletion) is a popular super-resolution fluorescence microscopy technique. In this paper, we present a concise guide to building a resonant-scanning STED microscope with ultrafast photon-counting acquisition. The STED microscope has two channels, using a pulsed laser and a continuous-wave (CW) laser as the depletion laser source, respectively. The CW STED channel preforms time-gated detection to enhance optical resolution in this channel. We use a resonant mirror to attain high scanning speed and ultrafast photon counting acquisition to scan a large field of view, which help reduce photobleaching. We discuss some practical issues in building a STED microscope, including creating a hollow depletion beam profile, manipulating polarization, and monitoring optical aberration. We also demonstrate a STED image enhancement method using stationary wavelet expansion and image analysis methods to register objects and to quantify colocalization in STED microscopy.

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