This technique exploits the fact that the measured photon distribution of a single fluorophore approaches the point spread function (PSF) of the microscope; thus, molecules can be localized to high precision by fitting to the ideal PSF. While this principle has been proposed before in connection high-resolution imaging, its utility was limited by the requirement that in order to be distinguished, molecules must be separated by at least the diffraction-limited resolution of the imaging instrument. This requirement has been met by using spectrally distinct, photobleaching, or blinking fluorophores. However, none of these methods has achieved a high density of emitters comparable to those possible with PALM, up to ~105/um2. This is possible by using photoactivatible xFP molecules, which can be activated, imaged, and photobleached over many cycles to reveal the positions of distinct sets of molecules. Multiple images are taken with total internal reflection fluorescence (TIRF) microscopy, and then combined to yield a single, high-resolution image with a high density of features. An image of the Golgi is reproduced below, to compare images from TIRF, PALM, and electron microscopy.
Figure: As published in 1, comparative TIRF (A) and PALM (B) images of the Golgi apparatus in a cryo-prepared thin section from a COS-7 cell expressing mEos-FP-tagged GalT. Higher magnification (C) of the box in (B) reveals a complex morphology not resolvable by TIRF, with thicker and thinner regions (arrows, C) similar to those seen in different cross-sectional TEM views of the Golgi apparatus (arrows, D) in a similarly prepared section.
1 E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess. (2006) Imaging Intracellular Fluorescent Proteins at Near-Molecular Resolution. Science 313:1642-45.