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.
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.
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.
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.
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.
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).
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.
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