Ultrahigh Resolution Imaging of Biomolecules by Fluorescence Photoactivation Localization Microscopy

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2009 Jun 3

PMID 19488720

Citations 16

Authors

Samuel T Hess

Travis J Gould

Mudalige Gunewardene

Joerg Bewersdorf

Michael D Mason

Affiliations

Soon will be listed here.

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.

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