Two-color CW STED nanoscopy

Xuanze Chen, Yujia Liu, Xusan Yang, Tingting Wang, Eric Alonas, Philip J. Santangelo, Qiushi Ren, Peng Xi

Research output: Chapter in Book/Report/Conference proceedingConference proceeding contributionpeer-review

2 Citations (Scopus)


Fluorescent microscopy has become an essential tool to study biological molecules, pathways and events in living cells, tissues and animals. Meanwhile even the most advanced confocal microscopy can only yield optical resolution approaching Abbe diffraction limit of ∼200 nm. This is still larger than many subcellular structures, which are too small to be resolved in detail. These limitations have driven the development of super-resolution optical imaging methodologies over the past decade. In stimulated emission depletion (STED) microscopy, the excitation focus is overlapped by an intense doughnut-shaped spot to instantly de-excite markers from their fluorescent state to the ground state by stimulated emission. This effectively eliminates the periphery of the Point Spread Function (PSF), resulting in a narrower focal region, or super-resolution. Scanning a sharpened spot through the specimen renders images with sub-diffraction resolution. Multi-color STED imaging can present important structural and functional information for protein-protein interaction. In this work, we presented a two-color, synchronization-free STED microscopy with a Ti:Sapphire oscillator. The excitation wavelengths were 532nm and 635nm, respectively. With pump power of 4.6 W and sample irradiance of 310 mW, we achieved super-resolution as high as 71 nm. Human respiratory syncytial virus (hRSV) proteins were imaged with our two-color CW STED for co-localization analysis.

Original languageEnglish
Title of host publicationSingle Molecule Spectroscopy and Superresolution Imaging VI
EditorsJörg Enderlein, Ingo Gregor, Zygmunt Karol Gryczynski, Rainer Erdmann, Felix Koberling
Place of PublicationBellingham, Washington
Number of pages7
Publication statusPublished - 2013
Externally publishedYes
EventSingle Molecule Spectroscopy and Superresolution Imaging VI - San Francisco, CA, United States
Duration: 2 Feb 20133 Feb 2013


OtherSingle Molecule Spectroscopy and Superresolution Imaging VI
Country/TerritoryUnited States
CitySan Francisco, CA


  • colocalization
  • super-resolution
  • Two-color CW STED


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