Stochastic optical reconstruction microscopy separates emitters in time rather than shrinking every illuminating spot. Switch only a sparse subset of labeled molecules into a fluorescent state, localize their individual point spread functions, switch or bleach them off, and repeat. Combining many fitted positions reconstructs structure finer than the individual diffraction-blurred spots.[ref]
Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) by Michael J. Rust and others. pp. 793–795, sparse activation and localization reconstruction
An interesting application is the neuronal axon. STORM revealed approximately – periodic organization of actin, spectrin and associated proteins, supporting a membrane-associated cytoskeletal architecture that ordinary fluorescence images did not resolve clearly.[ref]
Localization precision improves with detected photons, but reconstructed resolution also depends on label density, label size, drift and reliable separation of emitters. Many frames are needed, so motion and slow acquisition can limit live imaging. Multiple localizations of one blinking label must not automatically be counted as multiple molecules. A precise molecular fit and a faithful reconstructed biological structure are related but distinct achievements.
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