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Diffraction-Unlimited Fluorescence Imaging with an EasySTED Retrofitted Confocal Microscope

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Part of the Methods in Molecular Biology book series (MIMB,volume 1663)

Abstract

The easySTED technology provides the means to retrofit a confocal microscope to a diffraction-unlimited stimulated emission depletion (STED) microscope.

Although commercial STED systems are available today, for many users of confocal laser scanning microscopes the option of retrofitting their confocal system to a STED system ready for diffraction-unlimited imaging may present an attractive option. The easySTED principle allowing for a joint beam path of excitation and depletion light promises some advantages concerning technical complexity and alignment effort for such an STED upgrade. In the one beam path design of easySTED the use of a common laser source, either a supercontinuum source or two separate lasers coupled into the same single-mode fiber, becomes feasible. The alignment of the focal light distribution of the STED beam relative to that of the excitation beam in all three spatial dimensions is therefore omitted respectively reduced to coupling the STED laser into the common single-mode fiber. Thus, only minor modifications need to be applied to the beam path in the confocal microscope to be upgraded. Those comprise adding polarization control elements and the easySTED waveplate, and adapting the beamsplitter to the excitation/STED wavelength combination.

Key words

  • Super-resolution
  • Nanoscopy
  • Fluorescence microscopy
  • Confocal laser scanning microscopy
  • Time gating
  • Stimulated emission depletion

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Acknowledgments

This work was funded by the German Research Foundation (grant number 1850/30001355, www.dfg.de), and the Federal Ministry of Education and Research (“ALSComBi,” grant number 03IPT517Y, www.bmbf.de).

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Correspondence to André Klauss .

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Klauss, A., Hille, C. (2017). Diffraction-Unlimited Fluorescence Imaging with an EasySTED Retrofitted Confocal Microscope. In: Erfle, H. (eds) Super-Resolution Microscopy. Methods in Molecular Biology, vol 1663. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7265-4_4

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  • DOI: https://doi.org/10.1007/978-1-4939-7265-4_4

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7264-7

  • Online ISBN: 978-1-4939-7265-4

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