STED microscopy with continuous wave beams.
We report stimulated emission depletion (STED) fluorescence microscopy with continuous wave (CW) laser beams. Lateral fluorescence confinement from the scanning focal spot delivered a resolution of 29-60 nm in the focal plane, corresponding to a 5-8-fold improvement over the diffraction barrier. Axial spot confinement increased the axial resolution by 3.5-fold. We observed three-dimensional (3D) subdiffraction resolution in 3D image stacks. Viable for fluorophores with low triplet yield, the use of CW light sources greatly simplifies the implementation of this concept of far-field fluorescence nanoscopy.
Willig
Katrin I
KI
Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, 37077 Göttingen, Germany.
Harke
Benjamin
B
Medda
Rebecca
R
Hell
Stefan W
SW
D000818:Animals; D045744:Cell Line, Tumor; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D021621:Imaging, Three-Dimensional; D007150:Immunohistochemistry; D034882:Lamins / Q000032:analysis; D007834:Lasers; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D008863:Microspheres; D016900:Neurofilament Proteins / Q000032:analysis; D034881:Nuclear Lamina / Q000737:chemistry / Q000378:metabolism; D055095:Optics and Photonics; D016716:PC12 Cells; D050765:Qa-SNARE Proteins / Q000032:analysis; D051381:Rats; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D034882:Lamins; D016900:Neurofilament Proteins; D050765:Qa-SNARE Proteins