Watch one hour of TV, and you’re practically guaranteed to see at least one ad for the latest miracle stain remover. You might even see one for “stain resistant” fabrics. However, you’re not likely to see commercials for anything that promises to eliminate the risk of stains altogether.But a Harvard University team led by Xiaoliang Sunney Xie has found that stimulated Raman scattering (SRS) microscopy does just that for biomedical imaging: It allows researchers to watch real-time movement of molecules in live cells, all without relying on traditional fluorescent stains, which can damage tissue samples.A Harvard University team used SRS microscopy to observe omega-3 fatty acids entering living cells. Courtesy of Christian Freudiger, Harvard University. Avoiding damage is the big advantage to skipping the staining step. “In the case of metabolites, such as lipids, and drugs, the target molecules are smaller than the fluorescent labels,” said Christian Freudiger, a doctoral student in the physics department at the university. Freudiger worked with Xie and Wei Min, both of the department of chemistry and chemical biology, and others to develop the technique. “Thus, its attachment can change the biological function. Label-free imaging based on SRS allows us to study these molecules without perturbation, even in living cells and tissue.“In the case of brain imaging, traditional tissue stains can be toxic and often require freezing or fixing the tissue and are thus not suitable for minimally invasive optical biopsy. SRS microscopy allows us to generate label-free chemical contrast in fresh and untreated tissue.”Inelastic light scattering can be used to probe molecules optically because of their “very characteristic vibrations,” which can be identified spectroscopically, Freudiger said. But the signals in traditional spontaneous Raman scattering are too weak to produce the necessary vibrational contrast.“The achievement of our work was to incorporate stimulated Raman scattering as a contrast mechanism,” he said. “Instead of illuminating the sample with a single monochromatic laser beam, as in spontaneous Raman scattering, two beams coincide on the sample. If the difference frequency of the two beams matches the molecular vibration of the target molecule, the Raman signal is enhanced by orders of magnitude, by virtue of stimulated excitation of molecular vibrations.”Raster-scanning a laser through the sample allows the researchers to create an image. “For every pixel, an SRS experiment is carried out to determine the concentration of the target molecule,” he said. “Putting all the pixels together, a three-dimensional chemical map of the sample can be generated.”The team used SRS microscopy to watch retinoic acid molecules penetrate the skin. Retinoic acid commonly is used on acne and wrinkles. Courtesy of Christian Freudiger, Harvard University.Freudiger and his colleagues have used SRS microscopy in a range of applications, from observing omega-3 fatty acids as they enter live cells to watching retinoic acid, used on acne and wrinkles, as it penetrates the skin. They even have imaged individual neurons in the brains of mice.And the possibilities seem endless.“In principle, SRS can be used to image any type of ‘small molecule’ that cannot be fluorescently labeled,” Freudiger said. He added that current research includes imaging of neurotransmitters as well as metabolites such as adenosine triphosphate and adenosine diphosphate.“On the medical side,” he said, “we are hoping to develop SRS as an imaging technology that might eventually replace traditional [hematoxylin and eosin] staining and allow label-free in situ diagnosis.”