title: Resonant-scanning dual-color STED microscopy with ultrafast photon counting: A concise guide.
abstract: 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.
authors.affiliation: Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine, UCLA, United States; Cardiovascular Research Laboratory, David Geffen School of Medicine, UCLA, United States. Electronic address: ywu.thu@gmail.com.
authors.forename: Yong
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine, UCLA, United States.
authors.forename: Xundong
authors.initials: X
authors.lastname: Wu
authors.affiliation: Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine, UCLA, United States; Department of Molecular & Medical Pharmacology, David Geffen School of Medicine, UCLA, United States; Cardiovascular Research Laboratory, David Geffen School of Medicine, UCLA, United States.
authors.forename: Ligia
authors.initials: L
authors.lastname: Toro
authors.affiliation: Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine, UCLA, United States; Department of Physiology, David Geffen School of Medicine, UCLA, United States; Cardiovascular Research Laboratory, David Geffen School of Medicine, UCLA, United States.
authors.forename: Enrico
authors.initials: E
authors.lastname: Stefani
mesh_terms: D007089:Image Enhancement; D007834:Lasers; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D038761:Photobleaching
keywords: Colocalization; Fluorescence microscopy; Image analysis; Resonant scanning; STED microscopy