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

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

Author information

1
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.
2
Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine, UCLA, United States.
3
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.
4
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.

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.

KEYWORDS:

Colocalization; Fluorescence microscopy; Image analysis; Resonant scanning; STED microscopy

PMID:
26123183
PMCID:
PMC4630089
DOI:
10.1016/j.ymeth.2015.06.019
[Indexed for MEDLINE]
Free PMC Article

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