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STED (stimulated emission depletion) is a popular super-resolution fluorescence microscopy technique.
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In this paper, we present a concise guide to building a resonant-scanning STED microscope with ultrafast photon-counting acquisition.
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The STED microscope has two channels, using a pulsed laser and a continuous-wave (CW) laser as the depletion laser source, respectively.
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The CW STED channel preforms time-gated detection to enhance optical resolution in this channel.
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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.
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We discuss some practical issues in building a STED microscope, including creating a hollow depletion beam profile, manipulating polarization, and monitoring optical aberration.
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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.