SAN FRANCISCO — Researchers at Harvard University have developed a fluorescence microscopy technique that they claim can surpass the diffraction “blind spot” of conventional light microscopes.
The technique, known as stochastic optical reconstruction microscopy, or STORM, allowed the researchers to resolve the features of cells as small as 20 to 30 nm, an order of magnitude smaller than what is enabled by conventional fluorescence light microscopes.
This ability to directly visualize nanoscopic cellular structures and their spatial relationship in all three dimensions will greatly enhance scientists’ understanding of molecular processes in cells, according to the researchers.
Bo Huang, a postdoctoral fellow in the department of chemistry and chemical biology at Harvard University, and a co-developer of STORM, presented his group’s work in a poster at the 48th annual meeting of the American Society for Cell Biology, held here this week. The work also appeared online in the December issue of Nature Methods.
One of the major limitations of conventional light microscopes is that they are unable to resolve the images of two objects that are closer together than half the wavelength of light, which is, coincidentally, about the size of an organelle in the cell, Huang told CBA News. So the images produced are blurry and overlap no matter how high the magnification, and “you do not really see things clearly in the cell,” said Huang.
He explained that the objective of STORM microscopy is to overcome this limitation. One key concept is single-molecule characterization. “If you have one molecule in the sample, you can locate the center of the image,” said Huang.
The problem with a biological sample is that it often comprises hundreds of thousands of molecules that define the structure. However, in an image, these molecules overlap, so researchers cannot do simple localization to identify their center.
Fluorescence microscopy collects the fluorescent image from all of the molecules in the sample at the same time. Because all of the images of the molecules overlap, and the resolution of each image is determined by the diffraction of light, the entire image will have a resolution that is determined by the diffraction of light.
The other key concept of STORM is to use photoswitchable probes that can be switched between the visible state, which is fluorescent, and the invisible state, which is nonfluorescent, Huang said.
“With these so-called photoswitchable probes you can have most of the fluorophores in the invisible space, and then just activate a small fraction of them so that they are spatially resolvable.”
“With these so-called photoswitchable probes you can have most of the fluorophores in the invisible space, and then just activate a small fraction of them so that they are spatially resolvable,” said Huang. With these single-molecule images, the position of each activated fluorophore can be determined with high precision.
“You just keep repeating this procedure of activating fluorophores and localizing them until you get the position of coordinates of all the fluorophores in a sample. You can compile the coordinates to reconstruct a superresolution fluorescent image,” Huang explained.
In their Nature Methods paper, Huang and his colleagues reported that they were able to adapt STORM to combine 3D and multicolor imaging capabilities. They obtained whole-cell images with a spatial resolution of 20 to 30 nm and 60 to 70 nm in the lateral and axial dimensions, respectively.
The investigators imaged the entire mitochondrial network of fixed monkey kidney BS-C-1 cells and studied the spatial relationship between the mitochondria and microtubules. They reported that the 3D STORM images resolved mitochondrial morphologies and mitochondria-microtubule contacts that were obscured in conventional fluorescence images.
This technique is based on single-molecule localization of photoswitchable fluorophores and has been independently reported by different groups and given different names, including STORM and (fluorescence) photoactivation localization microcopy, or (f)PALM. Huang said that the only difference between these two techniques is the fluorophores used.
Eye of the STORM
Neither the ASCB poster nor the Nature Methods article mentioned the use of STORM in live-cell imaging. However, “other groups have demonstrated 20- to 30-second time resolution, and our group can also demonstrate a similar time resolution for imaging dynamic processes in the cell,” Huang said.
He added that the major framework of the STORM and (f)PALM techniques are mostly laid out in terms of single color-, multicolor-, 3D-, and live-cell imaging. “Right now another effort, at least in our group, is moving towards applications of STORM,” he said.
STORM and PALM would be applicable to drug discovery and development, said Huang. Many drugs act on membrane proteins and membrane receptors. For example, GPCRs are a big group of drug targets. There are many questions about how they are distributed, and how they are associated in the membrane. It is still very poorly understood, mainly because although “we postulate that there are membrane clusters of these receptors, we do not have the ability to see these clusters,” said Huang.
With this technique, especially as it is applied to live-cell imaging, researchers can gain a better understanding of how these membrane clusters function in the cell, and how drug candidates can affect or regulate their function in the cell, he said.
Huang said that he and his colleagues are working with companies to commercialize the STORM technology, but declined to comment further.