Changes in electron state of fluorescent indicators during photon excitation and emission (Jablonski profiles). Excitation (from S₀ to S₁) induced by 488 nm laser light (blue) requires one photon or by two-photon 800 nm light (red) requires two photons. After relaxation to the lowest energy levels, the reverse transition (from S₁ to S₀) releases a photon of longer wavelength (green). The incidence of photons at 592 nm (STED wavelength) induces the transition from S₁ to high levels of S₀ and suppression of fluorescence.