Abstract
Stimulated-emission depletion (STED) microscopy improves image resolution by encoding additional spatial information in a second stimulated-decay channel with a spatially-varying strength. Here we demonstrate that spatial information is also encoded in the fluorophore lifetime and that this information can be used to improve the spatial resolution of STED microscopy. By solving a kinetic model for emission in the presence of a time-varying STED pulse, we derive the effective resolution as a function of fluorophore lifetime and pulse duration. We find that the best resolution for a given pulse power is achieved with a pulse of infinitesimally short duration; however, the maximum resolution can be restored for pulses of finite duration by time-gating the fluorescence signal. In parallel, we consider time-gating in the presence of a continuous-wave (CW) STED beam and find that time-gating produces theoretically unbounded resolution with finite laser power. In both cases, the cost of this improved resolution is a reduction in the brightness of the final image. We conclude by discussing situations in which time-gated STED microscopy (T-STED) may provide improved microscope performance beyond an increase in resolution.
Highlights
In the past 10 years, several new microscopy techniques have demonstrated that far-field optics can be used to achieve nanometer-scale spatial resolution with visible light, breaking the resolution limit original proposed by Abbe [1,2,3,4]
We demonstrate that spatial information is encoded in the fluorophore lifetime and that this information can be used to improve the spatial resolution of Stimulated-emission depletion (STED) microscopy
By increasing the intensity of the STED illumination, the depleted region will increase, and the resulting point-spread functions (PSF) can be theoretically squeezed to arbitrarily small dimensions. (Throughout this Letter, we adopt the convention of judging spatial resolution by the spatial extent of the effective PSF.) Remarkably, STED microscopes based on this principle have achieved spatial resolutions up to ~6 nm starting with a PSF ~30-fold larger [13]
Summary
In the past 10 years, several new microscopy techniques have demonstrated that far-field optics can be used to achieve nanometer-scale spatial resolution with visible light, breaking the resolution limit original proposed by Abbe [1,2,3,4]. The core principle behind the resolution enhancement of STED is the ability to encode spatial information in the strength of a second decay channel This information is extracted by selectively discarding photons emitted via the second, stimulated channel from those emitted via spontaneous emission. We consider situations in which the STED beam is either pulsed or continuous wave (CW), and show that in both situations time-gating the fluorescence emission signal, i.e. discarding photons that arrive before a specific time, increases the spatial resolution of the final image. This increase in resolution is accompanied by a reduction in image brightness. We term this technique time-gated STED microscopy (T-STED) and conclude by discussing the potential benefits of this type of measurement
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