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Applied Physics B

, Volume 88, Issue 2, pp 161-165

Two-color far-field fluorescence nanoscopy based on photoswitchable emitters

  • H. BockAffiliated withDepartment of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry
  • , C. GeislerAffiliated withDepartment of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry
  • , C.A. WurmAffiliated withDepartment of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry
  • , C. von MiddendorffAffiliated withDepartment of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry
  • , S. JakobsAffiliated withDepartment of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry
  • , A. SchönleAffiliated withDepartment of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry
  • , A. EgnerAffiliated withDepartment of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry
  • , S.W. HellAffiliated withDepartment of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry Email author 
  • , C. EggelingAffiliated withDepartment of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry Email author 

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Abstract

We demonstrate two-color far-field fluorescence microscopy with nanoscale spatial resolution based on the photoswitching of individual fluorescent markers. By enabling, recording, and disabling the emission of the reversibly switchable fluorescent protein rsFastLime and of the organic fluorophore cyanine5, we recorded two-color nanoscale images inside whole cells. The position of individual emitters was determined with a typical accuracy of 20 nm, which largely constitutes the lateral resolution of the system. Photoswitching in two-color colocalization experiments represents a major step towards the application of far-field fluorescence nanoscopy to the study of (biological) samples on the macromolecular level.