New technique illuminates molecules with previously undetectable fluorescence (BioTechniques)
A team of researchers from Harvard University, led by chemist X. Sunney Xie, have developed a new microscopic technique that enables scientists to see molecules with undetectable fluorescence in color. Fluorescence is a phenomenon that occurs when an electron absorbs energy from light, and transitions to an excited state. The light energy is stored in a photon, which is released by the electron when the electron returns to its normal energy level. The energy contained in the released photon is emitted in wavelengths of detectable visible light that last only a few billionth of a second, called fluorescence.
Xie’s team studied molecules that are known for color, such as hemoglobin (the protein in red blood cells responsible for oxygen transport), that absorb light energy but do not fluoresce. The electrons in these molecules release the absorbed light energy by converting it to heat. “Since these molecules do not fluoresce, they have literally been overlooked by modern optical microscopes,” said Xie in a National Science Foundation (NSF) press release.
According to the NSF, to detect these non-fluorescent molecules, Xie’s team developed a novel type of microscopy based on stimulated emission. Stimulated emission occurs when an electron in an excited state, disturbed by a photon with a normal level of energy, drops to its normal state producing an additional photon to absorb its energy. According to the NSF, Xie’s technique generates and records a stimulated emission signal using timed input and output pulse trains. The intensity of the light energy in the input pulse train is modulated on and off at five MHz, to create a stimulated emission. Each pulse train has a duration of approximately 200 femtoseconds (10-15 seconds). The non-fluorescent molecules produce a signal that creates an image of these previously unseen molecules.
“There is no doubt that the study provides a unique way to image a wide range of molecules currently inaccessible to today’s state-of-the-art optical microscopes,” Zeev Rosenzweig, program director at the NSF’s Division of Chemistry, said in the press release. The Division of Chemistry contributed funding to Xie’s research.
Though the researchers reported that there is the potential for photo-damage, and that the cost of the system needs to be lowered before the technique could gain widespread use, the discovery is an important breakthrough.
According to the NSF, this research is important because current imaging technology, like an MRI or CT scan, do not have the spatial resolution needed to view tiny structures and require additional external contrast agents to create a viewable image. Xie’s technique is also a critical breakthrough because typical fluorescent labels, like green fluorescent protein, which are used to observe the activity of molecules, can disturb delicate biological pathways, especially when the protein is bigger than the molecule it is illuminating.
“This is just the beginning,” said Xie, in the press release. “Many interesting applications of this new imaging modality are forthcoming.” According to the NSF, one possible application for the new technique is mapping the delivery of non-fluorescent drugs to their target cells in color. The technique could also be used to view small structures like blood vessels or single capillaries.
The research was published in the Oct. 22 issue of Nature. Funding for the research was provided by the NSF Division of Chemistry and the U.S. Department of Energy’s Basic Energy Sciences Program.