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Related Experiment Video

Updated: Jun 25, 2026

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Published on: December 9, 2013

Imaging biological structures with fluorescence photoactivation localization microscopy.

Travis J Gould¹, Vladislav V Verkhusha, Samuel T Hess

¹Department of Physics and Astronomy and Institute for Molecular Biophysics, University of Maine, Orono, Maine 04469, USA.

Nature Protocols

|February 14, 2009

Summary

This summary is machine-generated.

Fluorescence photoactivation localization microscopy (FPALM) achieves super-resolution imaging of biological structures. This method precisely maps molecules using photoactivatable probes, enabling detailed cellular and nonbiological sample analysis.

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Published on: December 1, 2016

Area of Science:

Biophysics
Cell Biology
Microscopy

Background:

Fluorescence photoactivation localization microscopy (FPALM) offers subdiffraction-limited resolution for biological imaging.
It enables precise localization of photoactivatable probes to create high-resolution molecular maps.

Purpose of the Study:

To adapt a conventional widefield fluorescence microscope for FPALM.
To provide step-by-step procedures for obtaining and analyzing FPALM images.

Main Methods:

Utilizes repeated cycles of probe activation, readout, and bleaching.
Employs photoactivatable probes for precise molecular localization.
Adapts widefield fluorescence microscopy for super-resolution imaging.

Main Results:

Achieves effective resolution of tens of nanometers.
Enables imaging of membrane, cytoskeletal, and cytosolic proteins in fixed and living cells.
Allows quantification of molecular motions.
Applicable to both biological and nonbiological samples.

Conclusions:

FPALM provides a powerful tool for super-resolution cellular imaging.
The described protocol facilitates the application of FPALM and similar localization microscopy techniques.
Data acquisition and analysis are efficient, with acquisition in 1-30 min and analysis in 0.5-4 h.