Resonant-scanning dual-color STED microscopy with ultrafast photon counting: A concise guide.
Methods
; 88: 48-56, 2015 Oct 15.
Article
em En
| MEDLINE
| ID: mdl-26123183
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.
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MEDLINE
Assunto principal:
Aumento da Imagem
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Lasers
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Microscopia de Fluorescência
Idioma:
En
Ano de publicação:
2015
Tipo de documento:
Article