STED Nanoscopy with Time-Gated Detection: Theoretical and Experimental Aspects

Abstract

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.

Stimulated Emission Depletion Microscopy Resolves Individual Nitrogen Vacancy Centers in Diamond Nanocrystals

“…Spatially selective inhibition of the occupation of the first excited triplet state was induced by a doughnut-shaped STED laser beam (λ sted = 775 nm; pulse length, 2 ns; rep. rate, 8 MHz) at a maximal time averaged power of around 1 W. Fluorescence was detected in a time-gated fashion. 21,22 A time intensity autocorrelation detection unit enabled identification of NDs containing just a single NV center by means of their characteristic photon antibunching signature in the fluorescence intensity I(t) time autocorrelation function g (2) (τ) AEI(0)I(τ)ae/AEI(0)ae. 2,23À25 In the pulsed excitation regime, Poissonnian light yields photon correlation peaks that appear at every multiple of the inverse repetition rate of the laser.…”

A STED MICROSCOPE DESIGNED FOR ROUTINE BIOMEDICAL APPLICATIONS (Invited Paper)

“…Besides controlling the galvanometer scanners and the piezo stage for focus control, the FPGA adjusts the timing between the excitation and the STED laser pulses, and comprises a photon counting scheme with temporally gated detection [15,16]. For these purposes, programmable delay lines inside the Virtex V type FPGA chip are used which provide a time resolution of 80 ps.…”

A new filtering technique for removing anti‐Stokes emission background in gated CW‐STED microscopy

“…As an example, gated-STED (gSTED) microscopy has been developed. It is easy to demonstrate that the later the fluorescence signal of a fluorophore is observed, with respect to its excitation event, the more likely it becomes that it is inhibited if it is continuously exposed to stimulating photons [3,4]. In particular, measuring the fluorescence after a time T g , from the excitation events, ensures that the signal stems from fluorophores which have resided in the excited-state for at least a time T g and thereby, in the presence of the STED beam, have been exposed to stimulating photons for at least the same time.…”

“…Hence, this implementation, usually called gated CW-STED (gCW-STED) microscopy, provides subdiffraction resolution using moderate light intensity and thereby reduces photodamage effects [7]. Theoretically, the image contrast, thereby the effective spatial resolution, of a gCW-STED microscope can be continuously increased by delaying the detection [3,4], but the decrease in wanted signal caused by the time-gating imposes an upper limit on the choice of T g . In the presence of background, long T g can induce a strong reduction of the signalto-background ratio (SBR) which cancels out the benefits of time-gating and in the worst case reduces the effective resolution [4].…”