Resolution scaling in STED microscopy

Abstract

We undertake a comprehensive study of the inverse square root dependence of spatial resolution on the saturation factor in stimulated emission depletion (STED) microscopy and generalize it to account for various focal depletion patterns. We used an experimental platform featuring a high quality depletion pattern which results in operation close to the optimal optical performance. Its superior image brightness and uniform effective resolution <25 nm are evidenced by imaging both isolated and self-organized convectively assembled fluorescent beads. For relevant saturation values, the generalized square-root law is shown to predict the practical resolution with high accuracy.

Stimulated emission depletion-based raster image correlation spectroscopy reveals biomolecular dynamics in live cells

“…S3). The 100-fs pulse of the Ti:Sa laser is stretched to 200 ps by 60 cm of SF6 glass (AR680-800 SF6; Laser Components, Olching, Germany) followed by 100 m of polarization-maintaining single-mode fibre (PMJ-A3HPC; OZ Optics, Ottawa, Ontario, Canada) 20 . The long STED pulse ensures a complete coverage of the duration of the excitation pulse to efficiently suppress pre-STED fluorescence emission, and minimizes two-photon excitation by the depletion beam.…”

Comparing video‐rate STED nanoscopy and confocal microscopy of living neurons

“…If a molecule gets excited it is instantly pushed back to the ground state by stimulated emission effected by the STED beam. With increasing intensity of the STED beam, the area wherein the fluorophores are not turned off shrinks in principle to the size of a molecule [28,31]. With the STED beam blocked, the microscope simply operates as a standard confocal laser scanning microscope.…”

Frontiers in diffraction unlimited optical methods for spin manipulation, magnetic field sensing and imaging using diamond nitrogen vacancy defects

“…Moreover, the phononic coupling in the 3 A state enables quenching of the excited NV centres by stimulated emission more effi ciently [1,11] . A steep photoluminescence decay is observed when the STED beam with intensity I STED is applied to NV, yielding a saturation intensity of I S = 6.6 MW cm -2 , while typical dye fl uorescent beads have a I S = 11 MW cm -2 [62] . Since the fl uorescence is completely switched off when I STED > > 3 I S and the resolution increases with the ratio between the maximum STED beam intensity and the saturation intensity, clearly diamond provides the better property for the maximum resolution achievable.…”