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

Area of Science:

• Microscopy
• Optical Physics
• Biophysics

Background:

• Stimulated emission depletion (STED) microscopy enhances spatial resolution beyond the diffraction limit.
• Time-gated detection leverages the shrinking emission region in STED microscopy for improved resolution.

Purpose of the Study:

• To develop a theoretical framework and present experimental data characterizing the time evolution of the STED microscope's point-spread function.
• To illustrate the physical basis, benefits, and limitations of time-gated detection in both continuous wave (CW) and pulsed STED lasers.

Main Methods:

• Theoretical modeling of STED microscope point-spread function evolution.
• Experimental validation using time-gated detection with CW and pulsed STED lasers.
• Analysis of spatial frequency suppression and signal reduction associated with gating.

Main Results:

• Time-gated detection significantly suppresses low spatial frequencies in CW-STED nanoscopy, enhancing effective resolution.
• The improvement in effective resolution with gating is minimal for all-pulsed STED modalities.
• Signal reduction due to gating is the primary limitation for gated CW-STED nanoscopy.
• Time-gated detection mitigates the impact of fluorescence lifetime variations on STED resolution.

Conclusions:

• Gated CW-STED nanoscopy offers enhanced resolution primarily through low spatial frequency suppression, with signal loss as the main constraint.
• Time-gated detection provides a valuable tool for improving STED microscopy resolution, particularly in CW configurations.
• The method also reduces artifacts caused by fluorescence lifetime heterogeneity.