Portraits of Life, One Molecule at a Time Randall C Willis
Molecular microscopy is allowing researchers to probe beyond the diffraction barrier.
o monitor the activity of biological molecules within cells and thereby determine how cells function, researchers have come to rely largely on fluorescence microscopy. The repertoire of inorganic and organic fluorophore labels has grown, and image acquisition and analysis platforms have improved. Despite this progress, the inherent diffraction barrier of light (~200 nm) has limited the application of fluorescence microscopy to gross approximations of biomolecular position on cellular substructures, such as the Golgi apparatus and mitochondria. To look beyond this boundary, researchers have turned to other methods, such as electron microscopy (EM) and fluorescence correlation spectroscopy (FCS). Although EM studies have provided considerable information about cellular structure and function, experiments cannot be run under ambient conditions; this precludes the technique’s use in monitoring biomolecular changes over time. In addition, the expense of the instrumentation and the expertise required to run it have restricted its application. Similarly, FCS studies have provided knowledge about biomolecular behavior and interactions at the single-molecule level, but they generally do so in the absence of other cellular information. “Some people really want to see the structures in their context,” says Martin Hoppe at Leica Microsystems. “They really want to do structure analysis and not just single-molecule detection.”
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But by the same token, Iain Johnson at Invitrogen says, to fully characterize the molecular interactions that generate biological activity, information ultimately has to be obtained at the single-molecule level. He offers the example of imaging an ensemble of 100 ligands binding to 100 receptors. “You can imagine two extreme situations—one in which all 100 receptor/ligand interactions are equivalent in efficacy and another in which only one receptor/ligand interaction elicits a biological effect and the other 99 are unproductive or counteractive,” he says. “Without the capacity to image individual receptor/ligand complexes, these different eventualities can only be unraveled by methods that are either indirect or lack the vital spatial context provided by imaging methods.” To address these challenges, over the past several years researchers have developed optical microscopy methods that manipulate laser physics or fluorophore chemistry to break the 200nm diffraction barrier. These methods can be subdivided into discrete categories—e.g., confocal, interference, multiphoton, deconvolution—with a myriad of interrelations between the methods (1).
Breaking barriers Regardless of the manipulations involved, each method for subdiffraction imaging relies on a basic concept. Even though reM A R C H 1 , 2 0 0 7 / A N A LY T I C A L C H E M I S T R Y
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solving two or more labeled objects within the diffraction barrier is difficult or impossible, a single object can be precisely localized by determining the center of its emission pattern and using a Gaussian function to pinpoint its point spread function (PSF). A PSF can be made sharper, and therefore the location better defined, by collecting more photons and minimizing the noise factors (2). With these considerations in mind, Stefan Hell and colleagues at the Max Planck Institute for Biophysical Chemistry (Germany) relied on the physics of light interference to develop 4Pi microscopy in 2001. This technique improves resolution in the z axis of a sample to ~100 nm (3). Hoppe explains, “In 4Pi microscopy, you basically look at the specimen with two objectives which are opposite each other, so you are looking basically from both sides at the same spot. You combine the resolving power of two objectives, which increases the numerical aperture and resolution. It is like extending the numerical aperture of an objective to 4.” For their part, Paul Selvin and Ahmet Yildiz at the University of Illinois at Urbana–Champaign developed fluorescence imaging with 1-nm accuracy (known as FIONA). The technique enabled them to monitor the migration of single Cy3 or rhodamine molecules conjugated to myosin proteins as the proteins “walked” across actin strands on a glass slide in 40–80-nm increments (4). Selvin’s group also realized that two identical fluorophores attached to a sample do not photobleach at precisely the same time. They took advantage of this to develop single-molecule high-resolution imaging with photobleaching (SHRImP, 5). Essentially, the investigators continuously excite two fluorophores, generating an image that is the sum of the overlapping PSFs (mT). When one fluorophore photobleaches, the overall PSF decreases and shifts, which allows researchers to localize the second fluorophore (m2). By subtracting this information from the combined PSF, they can calculate the location of the other fluorophore (m1 = mT – m2). By this method, Selvin and colleagues could resolve individual fluorophores on Cy3-labeled DNA immobilized on glass slides down to a resolution of 10–20 nm. As a potential application for SHRImP, the researchers suggest high-density mapping of single nucleotide polymorphisms for large-scale genomics projects. Markus Sauer and colleagues at the University of Heidelberg and the University of Bielefeld (both in Germany) took SHRImP one step further by using two fluorescent dyes that had similar excitation wavelengths but different fluorescence lifetimes and emission maxima (6). By attaching the dyes to DNA molecules, the researchers imaged the molecules on slides with a combination of confocal spectrally resolved fluorescence-lifetime imaging microscopy and polarization-modulated excitation spectroscopy and achieved similar results. For Xiaowei Zhuang and colleagues at the Howard Hughes Medical Institute (HHMI) at Harvard University, however, the process of fluorophore interaction was too passive. They wondered what would happen if researchers could control the behavior of the fluorescent molecules. If only a subset of well1786
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separated fluorophores is excited, Zhuang and colleagues conjectured, the positions of each one can be determined precisely. A researcher can then turn the first set of fluorophores off and turn some of them on again. With this mission, they developed stochastic optical reconstruction microscopy (STORM, 7 ). This concept led to the use of photoswitchable fluorophores that allow the researcher to excite and quench the molecules stochastically. For STORM, Zhuang and colleagues used a Cy5–Cy3 fluorophore switch whereby the Cy5 molecule is activated by green light, with the help of Cy3, and deactivated by red light. Thus, researchers first switch a sample to the dark state and then excite a resolvable fraction of fluorophores. They then repeat the deactivation/activation cycle to determine the positions of the remaining fluorophores. By attaching Cy5–Cy3 switches to DNA, Zhuang’s group found they could accurately resolve switches 46 nm apart. Independently, Harald Hess and colleagues from HHMI’s Janelia Farm developed photoactivated localization microscopy (PALM), which relies on photoswitchable fluorescent proteins rather than dyes (8). Hess’s group first activated a population of fluorescent proteins and then excited the activated fluorophores. They acquired an image of the sample until the activated fluorophores were largely photobleached. The investigators went on to repeat the cycle several times until no further excitation was detected. By using PALM, Hess’s group imaged a variety of photoactivatable fusion proteins with green fluorescent protein (GFP) in thin cryosections of cultured mammalian cells and accurately determined the positions of proteins
