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Related Experiment Video

Updated: Jul 20, 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

Ultra-high resolution imaging by fluorescence photoactivation localization microscopy.

Samuel T Hess¹, Thanu P K Girirajan, Michael D Mason

¹Department of Physics and Astronomy, University of Maine, Orono, ME 04469, USA. sam.hess@umit.maine.edu

Biophysical Journal

|September 19, 2006

Summary

A new fluorescence imaging technique, Fluorescence Photoactivation Localization Microscopy (FPALM), achieves super-resolution by precisely localizing individual fluorescent molecules. This breakthrough overcomes the diffraction limit, enabling visualization of nanoscale biological structures.

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Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

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Last Updated: Jul 20, 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

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

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Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Published on: December 22, 2015

Area of Science:

Biophysics
Optical Microscopy
Nanotechnology

Background:

Visible light microscopy is limited by the diffraction limit, hindering the study of nanoscale biological structures.
Existing fluorescence imaging techniques struggle to resolve fine details in biological samples.

Purpose of the Study:

To develop a novel fluorescence imaging method that surpasses the classical diffraction limit.
To enable the visualization of spatial distributions of fluorescent molecules at the nanoscale.

Main Methods:

Developed Fluorescence Photoactivation Localization Microscopy (FPALM) using photoactivatable green fluorescent protein (PA-GFP).
Controlled fluorophore activation and photobleaching using specific laser intensities (405-nm for activation, Ar+ ion laser for excitation).
Localized individual fluorophores with high precision using CCD imaging and precise position determination.

Main Results:

Achieved localization precision up to 10-fold better than the diffraction limit.
Observed heterogeneities on length scales of tens of nanometers in PA-GFP on glass.
Demonstrated resolution significantly better than the diffraction limit, resolving features of approximately 86 nm on a sapphire crystal surface.

Conclusions:

FPALM overcomes the diffraction limit, offering unprecedented resolution in fluorescence imaging.
The technique allows for precise localization and counting of fluorescent molecules.
FPALM opens new avenues for investigating biological questions previously limited by microscope resolution.