Practice question

STED microscopy breaks diffraction by superimposing two synchronized lasers. A diffraction-limited excitation beam is overlapped by a doughnut-shaped depletion beam that drives stimulated emission at periphery, quenching fluorescence everywhere except central zero-intensity spot. This confined emissive region shrinks to 20-70 nm laterally, enabling super-resolution imaging of living cells. Unlike stochastic localization methods requiring extensive computational reconstruction, STED achieves resolution through targeted deterministic de-excitation, providing rapid scanning without heavy post-processing, ideal for observing synaptic vesicles, cytoskeletal filaments, and protein clusters in real time.