Optical molecular microscopy

In recent years, several techniques have been developed to beat the diffraction limit for optical microscopy. Two independent teams of researchers have now developed another diffraction-beating technique, based on a workhorse of modern cell biology—a fluorescing molecule, or fluorophore, that can be made to attach to a variety of targets in a cell. In the new technique, the fluorophores are photo-switchable; when dimly and briefly illuminated at an appropriate wavelength, only a few in the field of view are activated at any one time. The brightest ones can then be localized to within a few nanometers. By repeatedly imaging the same area and adding up many such sparse images, researchers build a composite that displays the entire field of view with near-molecular resolution. A group led by Eric Betzig (Howard Hughes Medical Institute [HHMI], Janelia Farm Research Campus, in Ashburn, Virginia) and Harald Hess (NuQuest Research LLC in La Jolla, California) calls the technique PALM, for photoactivated localization microscopy. A PALM image can be acquired in 2–12 hours. Shown here is a standard fluorescence microscopy image (top) of mitochondria in a frozen thin slice of a cell; the boxed area is enlarged in the PALM image below, in which more than 5500 molecules were localized. Meanwhile, a group led by Xiaowei Zhuang (Harvard and HHMI) calls the technique STORM, for stochastic optical reconstruction microscopy, and acquires complete images in minutes. Zhuang’s group uses fewer molecules than the other group but still resolves 10–20 of them within a normally unresolvable area. An additional complication Zhuang’s group solved involves using a fluorophore that can be switched on and off hundreds of times. (E. Betzig et al., Science, DOI:10.1126/science.1127344, 2006 ; M. J. Rust et al., Nature Methods, DOI:10.1038/nmeth929, 2006 .)