STED Nanoscopy: Beating the Diffraction Limit

Depletion Beam

Depletion power I / I_sat 20

Objective NA 1.40

Sample

Point-source separation 150 nm

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Diffraction limit d₀

– nm

STED spot d_STED

– nm

Enhancement

–×

Beam position

0 nm

How it works

Conventional (confocal) fluorescence microscopy cannot resolve two points closer than the Abbe diffraction limit, set by the emission wavelength λ and the objective's numerical aperture NA:

d₀ = 0.51 · λ / NA

STED microscopy beats this limit optically, without any post-processing. A normal diffraction-limited excitation spot switches on fluorophores, but a second, red-shifted "donut" beam — with a true intensity zero at its exact centre — is overlaid on it and forces every excited molecule outside that zero back to the ground state by stimulated emission before it can fluoresce. Only the vanishingly small region at the donut's null point is left free to emit spontaneously. Raising the depletion beam's peak intensity I relative to the fluorophore's saturation intensity I_sat squeezes that remaining "on" region down following:

d_STED = d₀ / √(1 + I / I_sat)

This simulator scans that effective spot across two fixed point emitters and plots the collected signal as it sweeps. At low depletion power the two emitters' broad diffraction-limited spots overlap into a single blurred peak; as I/I_sat increases, d_STED shrinks below the separation and the scan resolves two distinct peaks — the same trick Stefan Hell won the 2014 Nobel Prize in Chemistry for, now standard in commercial STED microscopes reaching ~20–50 nm resolution.

Depletion power — raises I/I_sat, shrinking the effective emitting spot (the bright core inside the orange donut ring).
Objective NA — sets the baseline diffraction limit d₀ that STED then improves on.
Separation — moves the two point sources; find the depletion power where they change from one merged peak to two.

Effective emission spot Fluorophore molecules Scan signal profile

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