What is the principle behind STED microscopy?
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, cytoskele
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.