STED nanoscopy with time-gated detection: theoretical and experimental aspects.
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.
Nanophysics, Istituto Italiano di Tecnologia, Genoa, Italy. giuseppe.vicidomini@iit.it
Giuseppe
G
Vicidomini
Andreas
A
Schönle
Haisen
H
Ta
Kyu Young
KY
Han
Gael
G
Moneron
Christian
C
Eggeling
Stefan W
SW
Hell
D004867:Equipment Design; D019544:Equipment Failure Analysis; D007089:Image Enhancement; D007834:Lasers; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*