How does stimulated emission depletion microscopy achieve superresolution?

Stimulated emission depletion (STED) microscopy achieves superresolution by bypassing the Abbe limit, which traditionally restricts the resolution of optical microscopes to approximately half the wavelength of the light used.12

The technique functions by treating a specimen with fluorescent dyes and utilizing two synchronized laser beams:12

• Excitation: The first laser beam excites the fluorophores in the sample.12
• Depletion: The second laser beam, which is modified to have zero intensity at the center of its focus, immediately returns the excited fluorophores in the surrounding area to the ground state, effectively turning off their fluorescence.12

By superimposing these beams, the system minimizes the effective area of illumination to a tiny region at the center of the focus. As these beams scan across the specimen, an image is gradually constructed with a resolution that can reach the single-nanometer range, allowing researchers to observe fine cellular details that were previously blurred by diffraction.12