Which super-resolution microscopy technique uses a depletion laser to confine excitation to a nanometer scale region?
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-pro
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.