PLoS ONE (Jan 2013)

STED nanoscopy with time-gated detection: theoretical and experimental aspects.

  • Giuseppe Vicidomini,
  • Andreas Schönle,
  • Haisen Ta,
  • Kyu Young Han,
  • Gael Moneron,
  • Christian Eggeling,
  • Stefan W Hell

DOI
https://doi.org/10.1371/journal.pone.0054421
Journal volume & issue
Vol. 8, no. 1
p. e54421

Abstract

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In a stimulated emission depletion (STED) microscope the region in which fluorescence markers can emit spontaneously shrinks with continued STED beam action after a singular excitation event. This fact has been recently used to substantially improve the effective spatial resolution in STED nanoscopy using time-gated detection, pulsed excitation and continuous wave (CW) STED beams. We present a theoretical framework and experimental data that characterize the time evolution of the effective point-spread-function of a STED microscope and illustrate the physical basis, the benefits, and the limitations of time-gated detection both for CW and pulsed STED lasers. While gating hardly improves the effective resolution in the all-pulsed modality, in the CW-STED modality gating strongly suppresses low spatial frequencies in the image. Gated CW-STED nanoscopy is in essence limited (only) by the reduction of the signal that is associated with gating. Time-gated detection also reduces/suppresses the influence of local variations of the fluorescence lifetime on STED microscopy resolution.