New highly sensitive, 3D microscopy technique enables real-time live cell study

DECEMBER 18, 2008--A new type of highly sensitive microscopy, based on stimulated Raman scattering (SRS), promises to substantially improve bio imaging by allowing scientists to see the location of metabolites and drugs in living cells and tissues. The method works without fluorescent labeling, which can perturb samples. Developed by the research group at Harvard University known for its work on CARS (coherent anti-Stokes Raman) microscopy, the new method is, "not only is more sensitive than CARS and orders of magnitude more sensitive than confocal Raman microscopy, but also provides SRS spectra that are identical to spontaneous Raman spectra," according to Professor Xiaoliang Sunney Xie, who led the research. The work is described this week in the journal Science by Xie, graduate student Christian W. Freudiger, and postdoctoral fellow Wei Min.

"SRS microscopy is a big leap forward in biomedical imaging, opening up real-time study of metabolism in living cells," says Xie, professor of chemistry and chemical biology in Harvard's Faculty of Arts and Sciences. "We've already used the technology to map lipids in a live cell, and to measure diffusion of medications in living tissue. These are just two early examples of how SRS microscopy may impact cell biology and medicine."

Xie, Freudinger, and Min's mapping of saturated and unsaturated fats in live cells offers exciting new opportunities for metabolic studies of omega-3 fatty acids, required but not produced by the human body. Despite a growing body of evidence suggesting that omega-3 fatty acids provide many health benefits such as dampening inflammation, lowering blood triglyceride levels, and killing cancer cells, almost nothing is known about how fats like omega-3 are actually processed by our bodies.

"Our diets have changed greatly in recent decades," Xie says. "As a unique technology capable of observing fat distribution in live cells -- and of differentiating between types of fat -- SRS microscopy could prove useful in helping understand and treat the growing imbalance of saturated and unsaturated fats in our diets."

SRS microscopy could also prove useful in neuroimaging, since neurons are coated with fatty myelin sheaths. The researchers' use of SRS microscopy to analyze skin tissue could also open new frontiers in drug development. Xie and colleagues used SRS microscopy to view how well retinoic acid, a topical acne medication, is absorbed into skin cells. They also used the technique to capture deep-skin penetration by dimethyl sulfoxide (DMSO), a compound added to many topical medications and ointments to enhance absorption.

Scientists currently use a variety of techniques to visualize biomolecules, but most have significant limitations that are sidestepped by SRS microscopy. A jellyfish protein first discovered in 1962, green fluorescent protein (GFP), is now used extensively as a label for observing the activity of biomolecules. GFP labeling provides sharp images, but the bulky protein can perturb delicate biological pathways, especially in cases where its heft overwhelms smaller biomolecules. Also, GFP's characteristic glow subsides with time, making it infeasible for long-term tracking.

Much like SRS microscopy, conventional infrared (IR) and Raman microscopies measure the vibrations of chemical bonds between atoms. But they are low-sensitivity imaging techniques, and require either desiccated samples or high laser power, which limits use in imaging live specimens. Coherent anti-Stokes Raman scattering (CARS) microscopy, a field pioneered by Xie's own group, cannot provide clear enough contrast for most molecules.

Xie, Freudiger, and Min's co-authors on the Science paper are Brian G. Saar, Sijia Lu, and Gary R. Holtom of Harvard's Department of Chemistry and Chemical Biology; Chengwei He and Jing X. Kang of the Department of Medicine at Massachusetts General Hospital and Harvard Medical School; and Jason C. Tsai of Pfizer Global Medical.

The work was funded by Boehringer Ingelheim Fonds, the Army Research Office, the National Institutes of Health, the U.S. Department of Energy, the National Science Foundation, the Bill and Melinda Gates Foundation, and Pfizer Global Medical.

More information:
Science magazine
Xie group at Harvard

Posted by Barbara G. Goode, [email protected].