Many types of modern biomedical microscopes use pulses of light aimed at chemical probes to image proteins, membranes, and cell structures. New understanding of biological processes within living tissues, such as metabolism and DNA repair, rely on the work researchers have done to bring miniscule features into focus. Their techniques include mastery of sophisticated instruments and software, as well as the development of genetically encoded fluorescent proteins, called fluorophores.
“As great as some of the current instrumentation is, much is limited by the physics of light and fluorophores,” explained George Patterson, Ph.D., investigator in the Section on Biophotonics at the National Institute of Biomedical Imaging and Bioengineering (NIBIB), part of the National Institutes of Health. “We are always bumping up against resolution limits.”
Patterson’s team is working to push the boundaries of super-microscopic resolution. In the Sept. 3, 2015, online issue of Nature Communications, they reported that a specialized technique, called two-step fluorescence microscopy, overcomes prior limits to improved image resolution for live cellular processes, particularly in thick tissue, such as tumors and skin.
Two-step fluorescence microscopy ultimately relies on the discovery in the 1960s of color emitting molecules originally isolated from jellyfish that can be genetically expressed in biological systems. Researchers perfected the technique for embedding colorful protein molecules into cells in the 1990s. Patterson encountered the technique as a Ph.D. student, when he used fluorescent proteins to study glucose metabolism in the pancreas. From there, his interest in fluorescence techniques expanded to developing them for general use in cell biology.
Fluorescent proteins are important for many super-resolution imaging techniques. Patterson’s team worked with a recently discovered fluorescent protein derived from coral, called Padron. It is distinctive on the one hand because it is photo-switchable—it can be turned on and off by laser pulses. It also is exceptional because it is activated in a two-step fluorescence process. The researchers used this capability for better control of the imaging process.
Specifically, Padron requires two separate blue light pulses—one that it absorbs, causing molecules to be switched on—or activated—and another to excite electrons in the molecules so they emit a fluorescent signal. It can be deactivated when exposed to violet light in order to repeat the two-step cycle. “The key here is we don’t activate all of the protein at once,” Patterson said, noting that the method avoids saturating the activation of Padron—as if burning it out. This approach allows for multiple coarse images that can be assembled into a fine high resolution image. The researchers showed that this process could more effectively detect slices of an object at different depths. They combined the image sections to generate single images of super resolution.
Patterson is hopeful that two-step imaging will be successfully incorporated in improving several other super-resolution imaging techniques. “You don’t need to buy more expensive equipment, but more expensive equipment might benefit from this as well,” he said. “Further development of this particular approach, in combination with other microscopy techniques, can push the envelope a little farther.”
Patterson’s team undertook this research in collaboration with members of the Section on High Resolution Optical Imaging, led by Hari Shroff, fellow NIBIB investigator. The team continues efforts to improve results with Padron, particularly to improved image contrast. Contrast is a critical optical parameter that must be adjusted to obtain detail that stands out against the background biological processes or against adjacent details.
“The contrast ratio is really important to get this to work,” Patterson said. “That’s one of the things that we’re trying to engineer in addition to getting the fluorophore to behave the same at any concentration. Our interest is in developing these probes to try to push them towards ideal behavior.”
Ingaramo M., York A.G., Andrade E.J., Rainey K., Patterson G.H.Two-photon-like microscopy with orders-of-magnitude lower illumination intensity via two-step fluorescence.
Nat Commu 2015 Sep 3;6:8184.