Conventional optics can achieve a diffraction-limited resolution of only about a half a wavelength of the source light, which means that conventional microscopes have a maximum resolution of about 200 nm. In contrast, a technique called photoactivated localization microscopy (PALM) can achieve as much as 100 times better resolution. Researchers recently enhanced the technique by making it dual-color, enabling it to provide more information about how biomolecules interact.Triple-label imaging combines dual-label photoactivated localization microscopy (PALM) techniques with standard epifluorescence, as seen here in an HFF-1 cell. A diffraction-limited epifluorescence image (upper right) of mCerulean-tagged actin (blue) overlaid with PALM images of Dronpa-tagged paxillin (green) and tdEos-tagged vinculin (red) shows adhesion complexes at the periphery of the cell aligned with the ends of actin bundles. An expanded view of the boxed region (center left) using PALM reveals parallel arrays of interwoven paxillin and vinculin aggregates along the length of each adhesion complex, as well as possibly nascent adhesion complexes consisting of adjacent paxillin and vinculin aggregates. A still further magnified view (lower right) indicates other examples of adjacent aggregates. The research was performed by Hari Shroff, a postdoctoral researcher at the Ashburn, Va.-based Janelia Farm Research Campus of the Howard Hughes Medical Institute, by other investigators at the institute, and by researchers from NIH in Bethesda, Md., and from Florida State University in Tallahassee.PALM produces its high resolution by using photoactivatable fluorescent proteins. Subsets of these proteins are rendered fluorescent when exposed to the proper wavelength of light and then are imaged. The proteins are far enough apart that their diffraction-limited images do not overlap, and each serves as a point source of light. Every photon collected enables a more precise determination of the location of each source. Tens of thousands of photons can be captured from each fluorescent label before it photobleaches completely. The location uncertainty of every fluorescent molecule can then potentially be reduced 100-fold.Optimizing resolutionHowever, achieving the maximum possible resolution takes the most time. Consequently, the practically achievable resolution will be less than the maximum.For PALM to work, the fluorophores must be sufficiently dense and significantly brighter than the background. If that is the case, the technique enables superresolution even when the fluorophores are very dense, an important attribute in biological applications where many proteins can interact to form functional units. Finally, each fluorophore must produce enough photons, typically around 1000 or so, before disappearing from view. Researchers have achieved superresolution in two colors via dual-label photoactivated localization microscopy. They expose a specimen initially expressing inactive EosFP and Dronpa molecules (step 1) to 405-nm activation light and 561-nm Eos-excitation light (steps 2 and 3) until all EosFP molecules are detected, localized and bleached (step 4). As a result, many of the Dronpa molecules are activated (step 4). They then deactivate these molecules using an intense 488-nm light (step 5). Finally, they apply both 405- and 488-nm light (steps 6 and 7) to serially activate, detect, localize and eventually bleach (step 8) all remaining Dronpa molecules. Images encompassing 100,000 to as many as 1 million molecules are acquired, typically in five to 30 min. Images reprinted with permission of PNAS. Extending this idea to dual colors requires appropriate photoactivatable fluorescent protein pairs -- not a trivial task, Shroff said. The pairs must be spectrally distinct so that molecules of one type can be distinguished from those of the other. However, current photoactivatable fluorescent proteins fall into two classes. One emits green fluorescence when turned on, and the other shifts from green to orange upon activation. Thus, the latter type provides a bright background that hinders the isolation and localization of the first.The researchers solved this problem by using Dronpa, a reversible photoactivatable fluorescent protein that emits in the green. They paired this with EosFP, which shifts from green to orange when photoactivated. They started with a specimen expressing inactive EosFP and Dronpa. They activated the EosFP using a 405-nm source and excited it with a 561-nm source, repeating the activation and excitation process until all of the EosFP molecules were located and photobleached.This left them with a large pool of activated green Dronpa molecules, so they subjected the sample to 488-nm light to cause the Dronpa to revert to an inactivated state. They activated the Dronpa molecules with a 405-nm beam and excited them with a 488-nm source, again repeating the process over thousands of images until the second fluorescent protein photobleached into invisibility.Multicolor superresolution has already been demonstrated with other techniques. However, these utilize fluorescent tags introduced via antibodies or biotinylation. The relationship between these exogenous tags and the target adds 10 to 20 nm of spatial uncertainty. The endogenous tags in the PALM method do not suffer from this problem. They also can be used for conventional microscopy, thereby providing additional useful information. In a demonstration setup, the researchers used an Olympus microscope and an electron-multiplying CCD camera from Andor Technology of Belfast, UK. The illumination system consisted of a 561-nm laser from CrystaLaser of Reno, Nev., a 488-nm laser from the Mountain View, Calif.-based Spectra-Physics Lasers Div. of Newport Corp. and a 405-nm laser from Coherent of Santa Clara, Calif. The three beams were combined using lenses, filters, dichroic mirrors and other optical elements and then fed into the microscope. With this setup, the researchers could image a whole fixed cell with 20- to 30-nm resolution in five to 30 min. The work was published in the Dec. 18 issue of PNAS.The researchers examined endogenously expressed Dronpa/EosFP in HFF-1 cell adhesion complexes, the central attachment points between the cytoskeleton and the substrate in migrating cells. These complexes can be as small as 0.5 μm and result from the interaction of more than 90 different proteins. The researchers found these complexes, which were fuzzy blobs when viewed conventionally, to be distinct interlocking nanoaggregates when imaged with PALM. The images revealed an interwoven arrangement, suggesting an interactive organization of molecules.“This is a novel finding, and we did not expect it. We are excited by this because we think it points out the potential of dual-label PALM for new biological findings,” Shroff said. These nanoaggregates, the researchers noted, could be caused by an artifact introduced during cell fixation. However, preliminary work on nonfixed cells indicates that that is not the case. If the results hold up, it may be necessary to rethink the idea of colocalization represented by these complexes.The researchers also performed three-color imaging, combining dual-label PALM with the fluorescent protein mCerulean. The latter indicated the location of actin fibers in the cell.Work in the area of PALM imaging continues, with live-cell and three-dimensional efforts under way. Shroff noted that there has been good progress on the former. There also are commercialization prospects as a result of an agreement signed late last year with Carl Zeiss MicroImaging of Jena, Germany. In addition, the current dual-label approach is sequential in nature, with one label imaged and then the other, but that may change in the future.