Practice question

STED, stimulated emission depletion, breaks the diffraction limit by superimposing a second doughnut-shaped depletion laser over the excitation spot. The first laser excites fluorophores in a diffraction-limited volume, while the red-shifted high-intensity STED beam forces excited molecules in the periphery back to ground state via stimulated emission before spontaneous fluorescence occurs. Only molecules at the zero-intensity centre remain fluorescent, shrinking effective point spread function to 30-50 nanometers. Scanning this narrowed spot builds super-resolved images of synapses, cytoskeletal filaments and nuclear pores beyond Abbe limit.