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Author Notes:

E-mail: xipeng@pku.edu.cn

Conceived and designed the experiments: PX QR DJ JAP.

Performed the experiments: YL YD EA WZ.

Analyzed the data: YL PX.

Contributed reagents/materials/analysis tools: EA PJS WZ JT.

Wrote the paper: YL PJS DJ PX.

The authors would like to acknowledge Dr. Katrin Willig, Dr. Benjamine Harke and Dr. Haisen Ta for technical instructions and providing the gold nanoparticle and intermediate filament samples; and Dr. Andreas Schönle for helping with Imspector.

PX thank Prof. Stefan W. Hell for mentoring and training on STED nanoscopy instrumentation.

Part of this research is sponsored by Olympus Australia, however this does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.

Subjects:

Research Funding:

This research is supported by the “973” Major State Basic Research Development Program of China (2011CB809101, 2010CB933901, 2011CB707502), the National Natural Science Foundation of China (61178076), PKU-GT/Emory University BME Seed Grant funded by Wallace H. Coulter Foundation, and seed fund support from Olympus Australia.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Keywords:

  • Science & Technology
  • Multidisciplinary Sciences
  • Science & Technology - Other Topics
  • MULTIDISCIPLINARY SCIENCES
  • STIMULATED-EMISSION-DEPLETION
  • OPTICAL RECONSTRUCTION MICROSCOPY
  • FLUORESCENCE MICROSCOPY
  • 2-PHOTON EXCITATION
  • SINGLE-WAVELENGTH
  • BREAKING
  • REVEALS
  • EVENTS
  • STORM
  • LIMIT

Achieving lambda/10 Resolution CW STED Nanoscopy with a Ti:Sapphire Oscillator

Tools:

Journal Title:

PLoS ONE

Volume:

Volume 7, Number 6

Publisher:

, Pages e40003-e40003

Type of Work:

Article | Final Publisher PDF

Abstract:

In this report, a Ti:Sapphire oscillator was utilized to realize synchronization-free stimulated emission depletion (STED) microscopy. With pump power of 4.6 W and sample irradiance of 310 mW, we achieved super-resolution as high as 71 nm. With synchronization-free STED, we imaged 200 nm nanospheres as well as all three cytoskeletal elements (microtubules, intermediate filaments, and actin filaments), clearly demonstrating the resolving power of synchronization-free STED over conventional diffraction limited imaging. It also allowed us to discover that, Dylight 650, exhibits improved performance over ATTO647N, a fluorophore frequently used in STED. Furthermore, we applied synchronization-free STED to image fluorescently-labeled intracellular viral RNA granules, which otherwise cannot be differentiated by confocal microscopy. Thanks to the widely available Ti:Sapphire oscillators in multiphoton imaging system, this work suggests easier access to setup super-resolution microscope via the synchronization-free STED.

Copyright information:

© 2012 Liu et al.

This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
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