Localization Microscopy with Active Control

  • Barry R. MastersEmail author
Part of the Springer Series in Optical Sciences book series (SSOS, volume 227)


How does localization microscopy achieve superresolution? What do we mean by the word “localization”? The answer to these questions is the content of this chapter. First, I define some terms. Localization refers to the technique of locating the centroid or geometric center of the point spread function (PSF) of a fluorescent molecule. The localization of a molecule can be obtained with higher precision than resolution; precision increases with larger numbers of detected photons and with decreasing background fluorescence. Resolution (Chapter  2) refers to the smallest distance that separates two objects for which the two objects are imaged as two distinct objects. Photoswitching fluorescent molecules is a reversible or irreversible process in which a photon can alter the emission wavelengths of the molecule (e.g., from green fluorescent to red fluorescent). Photoactivation is the process in which molecules, which are nonfluorescent (in the OFF state) before they are activated with blue or ultraviolet light, exhibit normal absorption and emission spectra (in the ON state). Reversible photoactivated fluorescent molecules can cycle between the nonfluorescent OFF state and the fluorescent ON state many times; irreversible photoactivated fluorescent molecules can only be activated to the fluorescent state once. Superresolution microscopy achieves imaging of object details with a resolution that exceeds diffraction-limited resolution.


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© Springer Nature Switzerland AG 2020

Authors and Affiliations

  1. 1.Previously, Visiting Scientist Department of Biological EngineeringMassachusetts Institute of TechnologyCambridgeUSA
  2. 2.Previously, Visiting Scholar Department of the History of ScienceHarvard UniversityCambridgeUSA

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