Process
STED functions by depleting specific regions of the sample while leaving a center focal spot active to emit fluorescence. This focal area can be engineered by altering the properties of the pupil plane of the objective lens. The most common early example of this diffractive optical elements, or DOEs, is a doughnut shape used in two dimensional lateral confinement shown below. The red zone is depleted, while the green spot is left active. This DOE is generated by a circular polarization of the depletion laser, combined with a helical phase ramp. The lateral resolution of this DOE is typically between 30 and 80 nm. However, values down to 2.4 nm have been reported. Using different DOEs, axial resolution on the order of 100 nm have been demonstrated. A modified Abbe’s equation describes this sub diffraction resolution as: D=λ/(2nsinα*sqrt(1+I/Isat)). To optimize the effectiveness of STED, the destructive interference in the center of the focal spot needs to be as close to perfect as possible. That imposes certain constraints on the optics that can be used.
Read more about this topic: STED Microscopy
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