Imaging biological structures with fluorescence photoactivation localization microscopy

Abstract

Fluorescence photoactivation localization microscopy (FPALM) images biological structures with subdiffraction-limited resolution. With repeated cycles of activation, readout and bleaching, large numbers of photoactivatable probes can be precisely localized to obtain a map (image) of labeled molecules with an effective resolution of tens of nanometers. FPALM has been applied to a variety of biological imaging applications, including membrane, cytoskeletal and cytosolic proteins in fixed and living cells. Molecular motions can be quantified. FPALM can also be applied to nonbiological samples, which can be labeled with photoactivatable probes. With emphasis on cellular imaging, we describe here the adaptation of a conventional widefield fluorescence microscope for FPALM and present step-by-step procedures to successfully obtain and analyze FPALM images. The fundamentals of this protocol may also be applicable to users of similar imaging techniques that apply localization of photoactivatable probes to achieve super-resolution. Once alignment of the setup has been completed, data acquisitions can be obtained in approximately 1–30 min and analyzed in approximately 0.5–4 h.

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Figure 1: Concept of FPALM.
Figure 2: FPALM experimental setup.
Figure 3: Identifying single molecules.
Figure 4: Example timing sequences for FPALM acquisitions.
Figure 5: Typical FPALM image of Dendra2-actin expressed in a fixed mouse fibroblast.
Figure 6: Illustration of troubleshooting of pixelization artifact in FPALM image of Dendra2-actin expressed in a fixed fibroblast.

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Acknowledgements

We thank Christopher Fang-Yen, Paul Blank, Joerg Bewersdorf, Joshua Zimmerberg, George Patterson, Julie Gosse and Michael Mason for useful discussions, Paul Millard and Carol Kim for use of equipment and reagents, Ed Allgeyer, Manasa Gudheti and Siyath Gunewardene for laboratory assistance, Matthew Parent for programming assistance and Thomas Tripp, Tony McGinn and Kyle Jensen for machining services. This work was supported by grants K25-AI-65459 from the National Institute of Allergy and Infectious Diseases (S.T.H.), CHE-0722759 from the National Science Foundation (S.T.H.), start-up funds from the University of Maine (S.T.H.) and by grants GM070358 and GM073913 from the National Institute of General Medical Sciences (V.V.V.).

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Correspondence to Samuel T Hess.

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Gould, T., Verkhusha, V. & Hess, S. Imaging biological structures with fluorescence photoactivation localization microscopy. Nat Protoc 4, 291–308 (2009). https://doi.org/10.1038/nprot.2008.246

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