Super-resolution microscopy is revolutionizing the ability to probe subcellular features as small as single molecules. Surpassing traditional limitations of conventional optical microscopes, these new technologies permit very precise visualization and measurement of features that are below the limits of diffraction.
The super-resolution toolbox includes localization microscopy that identifies the location of individual fluorophores a few at a time, structured illumination microscopy that allows a three-dimensional view, and hyperspectral confocal imaging, among others. Applications for the new technologies range from depicting the fine architecture of cellular immune processes to creation of improved biofuels.
Pavel Tolar, Ph.D., program leader in the division of immune cell biology at MRC National Institute for Medical Research, provides a perspective on several current imaging technologies.
“Traditional fluorescence microscopy is hampered by the resolution limit set by the diffraction of light that is ~200–300 nanometers. Recently, a number of novel instrument-based fluorescence approaches have been employed to circumvent these limitations. We are now seeing imaging down to a resolution that approaches molecular scale (10–15 nm).”
An example of such nanomolar-scale imaging is that of fluorescent resonance energy transfer (FRET), a single molecule technology in which energy is transferred from an excited molecular fluorophore (the donor) to another fluorophore (the acceptor).
“FRET is well-suited to detect protein interactions as well as conformational changes. This provides a remarkable visualization of dynamic protein function to monitor protein-protein interactions (e.g., how signaling complexes assemble) where the distance of interaction is from 2–10 nm. For that reason it is useful for drug screening. But, it is also limited by its low throughput.”
According to Dr. Tolar, the most accessible of these new technologies are photo-activated localization microscopy (PALM) and the related stochastic optical reconstruction microscopy (STORM).
“PALM/STORM are based on the detection and very precise localization of individual molecules. Both employ photo-activatable fluorescent labels for localizing many molecules in a sequential manner using repetitive cycles of activation and imaging. The end result is generation of a high-resolution image that maps the positions of all the molecules monitored. The technology currently is only limited by the number of molecules that can be activated at one time, their brightness, and the rate of photobleaching.”