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

Methods. 2015 Oct 15;88:48-56. doi: 10.1016/j.ymeth.2015.06.019. Epub 2015 Jun 27.


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.

Keywords: Colocalization; Fluorescence microscopy; Image analysis; Resonant scanning; STED microscopy.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Image Enhancement*
  • Lasers*
  • Microscopy, Fluorescence / instrumentation*
  • Photobleaching