Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy (FPALM)

¹ University of Maine

^* To whom correspondence should be addressed. E-mail: sam.hess{at}umit.maine.edu.

Submitted on June 12, 2006
Revised on July 11, 2006
Accepted on 28 August 2006

	Abstract

Biological structures span many orders of magnitude in size, but far-field visible light microscopy suffers from limited resolution. A new method for fluorescence imaging has been developed that can obtain spatial distributions of large numbers of fluorescent molecules on length scales shorter than the classical diffraction limit. Fluorescence photoactivation localization microscopy (FPALM) analyzes thousands of single fluorophores per acquisition, localizing small numbers of them at a time, at low excitation intensity. In order to control the number of visible fluorophores in the field of view and ensure that optically active molecules are separated by much more than the width of the point spread function (PSF), photoactivatable fluorescent molecules are used, in this case the photoactivatable green fluorescent protein (PA-GFP). For these photoactivatable molecules, the activation rate is controlled by the activation illumination intensity; non-fluorescent inactive molecules are activated by a high-frequency (405 nm) laser and are then fluorescent when excited at a lower frequency. The fluorescence is imaged by a CCD camera, and then the molecules are either reversibly inactivated or irreversibly photobleached to remove them from the field of view. The rate of photobleaching is controlled by the intensity of the laser used to excite the fluorescence, in this case an Ar+ ion laser. Because only a small number of molecules are visible at a given time, their positions can be determined precisely; with only ~100 detected photons per molecule, the localization precision can be as much as ten-fold better than the resolution, depending on background levels. Heterogeneities on length scales of order tens of nanometers are observed by FPALM of PA-GFP on glass. FPALM images are compared with images of the same molecules by widefield fluorescence. FPALM images of PA-GFP on a terraced sapphire crystal surface were compared with atomic force microscopy and show the FWHM of features ~86 ± 4 nm is significantly better than the expected diffraction limited optical resolution. The number of fluorescent molecules and their brightness distribution have also been determined using FPALM. This new method suggests a means to address a significant number of biological questions that had previously been limited by microscope resolution.

Key Words: nanoscale, photoactivatable green fluorescent protein, photoconversion, programmable molecules, single molecules

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