Enhanced lateral resolution in continuous wave stimulated emission depletion microscopy using tightly focused annular radially polarized excitation beam

J Biophotonics. 2019 Sep;12(9):e201900060. doi: 10.1002/jbio.201900060. Epub 2019 May 28.

Abstract

The lateral resolution of continuous wave (CW) stimulated emission depletion (STED) microscopy is enhanced about 12% by applying annular-shaped amplitude modulation to the radially polarized excitation beam. A focused annularly filtered radially polarized excitation beam provides a more condensed point spread function (PSF), which contributes to enhance effective STED resolution of CW STED microscopy. Theoretical analysis shows that the FWHM of the effective PSF on the detection plane is smaller than for conventional CW STED. Simulation shows the donut-shaped PSF of the depletion beam and confocal optics suppress undesired PSF sidelobes. Imaging experiments agree with the simulated resolution improvement.

Keywords: PSF engineering; STED microscopy; fluorescence microscopy; superresolution.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / chemistry
  • Algorithms
  • Animals
  • Cell Line, Tumor
  • Computer Simulation
  • Fluorescent Dyes / pharmacology
  • Humans
  • Light
  • Mice
  • Microscopy, Confocal / instrumentation*
  • Microscopy, Confocal / methods*
  • Microscopy, Fluorescence / instrumentation*
  • Microscopy, Fluorescence / methods*
  • Microtubules / chemistry
  • NIH 3T3 Cells
  • Normal Distribution

Substances

  • Actins
  • Fluorescent Dyes