Tuning donut profile for spatial resolution in stimulated emission depletion microscopy

Abstract

In stimulated emission depletion (STED)-based or up-conversion depletion-based super-resolution optical microscopy, the donut-shaped depletion beam profile is of critical importance to its resolution. In this study, we investigate the transformation of the donut-shaped depletion beam focused by a high numerical aperture (NA) microscope objective, and model STED point spread function (PSF) as a function of donut beam profile. We show experimentally that the intensity profile of the dark kernel of the donut can be approximated as a parabolic function, whose slope is determined by the donut beam size before the objective back aperture, or the effective NA. Based on this, we derive the mathematical expression for continuous wave (CW) STED PSF as a function of focal plane donut and excitation beam profiles, as well as dye properties. We find that the effective NA and the residual intensity at the center are critical factors for STED imaging quality and the resolution. The effective NA is critical for STED resolution in that it not only determines the donut shape but also the area the depletion laser power is dispersed. An improperly expanded depletion beam will have negligible improvement in resolution. The polarization of the depletion beam also plays an important role as it affects the residual intensity in the center of the donut. Finally, we construct a CW STED microscope operating at 488 nm excitation and 592 nm depletion with a resolution of 70 nm. Our study provides detailed insight to the property of donut beam, and parameters that are important for the optimal performance of STED microscopes. This paper will provide a useful guide for the construction and future development of STED microscopes.

Figure 1

Schematic of the construction of the CW STED microscope. The excitation was provided by an Ar ion laser (488 nm) and the depletion was from a 592 nm fiber laser. BPF: bandpass filter; VPP: vortex phase plate; L: lenses; P: polarizer; QWP: quarterwave plate; DCSP: dichroic mirror (short pass); NF: notch filter; DCLP: dichroic mirror (long pass); PH: pinhole; APD: avalanche photodiode detector.

Figure 2

Donut profile as a funciton of the depletion beam size. The depletion beam was first passed throught the vortex phase plate to form a donut-profiled beam. The donut beam was then collimated to have a beam diameter of (a) ∼6 mm and (b) ∼3 mm.

Figure 3

Donut profile as a funciton of the depletion beam polarization. (a) Right-handed circular as defined from the point of view of the receiver; (b) linear; and (c) left-handed circular. The vortex phase plate generates a right-handed spiral phase delay viewed from the receiver.

Figure 4

The effect of the center profile of the donut on STED images. (a) Assumed donut profile with the center portion filled to different levels of the maximum intensity; (b) simulated STED resolution at different filled levels; (c) simulated maximum STED image intensity at corresponding filled levels; and (d) resolution-depletion power relationship at different filled levels. The 340 nm donut was used in the simulation. The excitation and depletion laser powers were assumed to be 15 μW and 1.0 W, respectively.

Figure 5

Other factors affecting STED resolution. Simulated spatial resolution by varying (a) dye fluorescence lifetime; and (b) excitation beam width. The 340 nm donut was used in the simulation. The excitation and depletion laser powers were assumed to be 15 μW and 1 W, respectively.

Figure 6

Confocal and STED point spread function. (a) and (b) Confocal and STED image of 45 nm nanoparticles immobilized on glass surface, respectively; (c) intensity profiles across the images and their Lorentzian fitting of a particle in (a) and (b).

Figure 7

Confocal and STED images of 200 nm nanoparticles immobilized on the water/glass interface in the presence of a blanket of 1-fluoroheptane. (a) Confocal image; (b) original STED image.