Principles and Applications of Localization-Based Superresolution Microscopy (FPALM)
Sam Hess, University of Maine, Orono, USA

Diffraction limits the resolution that can be obtained with visible light microscopy to ~200 nanometers. However, much of biology occurs on much smaller (molecular) length scales. Several super-resolution light microscopy techniques have been invented to break the diffraction barrier by utilizing the principles of observation volume confinement, modulated illumination, and single molecule localization. Fluorescence photoactivation localization microscopy (FPALM) is an example of such a super-resolution light microscopy technique which can image living or fixed biological samples with demonstrated resolution of tens of nanometers, roughly an order of magnitude improvement over the diffraction limit. FPALM and similar methods use repeated cycles of photoactivation, localization, and photobleaching of many sparse subsets of photoactivatable molecules. The positions of single molecules can be determined (localized) with a precision better than the diffraction limited resolution. Final images are rendered by plotting the positions and intensities of thousands of localized molecules, yielding a map of the distribution of those molecules with an effective resolution limited by the localization precision and labeling density. These methods are now being used to address biological problems which were completely out of the question just a few years ago. Thus, their application to biological systems offers great opportunity for new insight.

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