Ultrahigh resolution imaging of biomolecules by fluorescence photoactivation localization microscopy.
Document Type
Article
Publication Date
2009
Keywords
Data-Interpretation-Statistical, Fluorescence-Recovery-After-Photobleaching, Fluorescent-Dyes, Microscopy-Fluorescence, Photobleaching, Photochemical-Processes, Proteins
First Page
483
Last Page
522
JAX Location
see Reprint Collection (a pdf is available)
JAX Source
Methods Mol Biol 2009; 544:483-522.
Abstract
Diffraction limits the biological structures that can be imaged by normal light microscopy. However, recently developed techniques are breaking the limits that diffraction poses and allowing imaging of biological samples at the molecular length scale. Fluorescence photoactivation localization microscopy (FPALM) and related methods can now image molecular distributions in fixed and living cells with measured resolution better than 30 nm. Based on localization of single photoactivatable molecules, FPALM uses repeated cycles of activation, localization, and photobleaching, combined with high-sensitivity fluorescence imaging, to identify and localize large numbers of molecules within a sample. Procedures and pitfalls for construction and use of such a microscope are discussed in detail. Representative images of cytosolic proteins, membrane proteins, and other structures, as well as examples of results during acquisition are shown. It is hoped that these details can be used to perform FPALM on a variety of biological samples, to significantly advance the understanding of biological systems.
Recommended Citation
Hess ST,
Gould TJ,
Gunewardene M,
Bewersdorf J,
Mason MD.
Ultrahigh resolution imaging of biomolecules by fluorescence photoactivation localization microscopy. Methods Mol Biol 2009; 544:483-522.