Enhanced CARS Enables High-Speed Imaging of Tissues

An enhanced spectroscopy technique for analyzing biological cells and tissues delivers stronger signals than those attained through conventional practices.

A team from the National Institute of Standards and Technology, in conjunction with the Cleveland Clinic, has demonstrated the improved method based on characteristic molecular vibration signatures.

A false-color BCARS image of mouse liver tissue picks out cell nuclei in blue, collagen in orange and proteins in green. The image shows an area about 200 µm across. Images Courtesy of Dr. Charles Camp Jr./NIST.

The technique, called broadband CARS (BCARS), is essentially an advanced form of coherent anti-Stokes Raman scattering. It can deliver signals 10,000 times stronger than those obtained through spontaneous Raman scattering and is also 100 times stronger than comparable coherent Raman scattering techniques, using a much larger portion of the vibrational spectrum.

The technique accesses the same spectral region in which existing coherent Raman methods obtain useful signals containing approximately five peaks with information about carbon-hydrogen and oxygen-hydrogen bonds.

However, the improved method can also pick up signals from the "fingerprint" spectral region, which has approximately 50 peaks and contains most of the useful molecular identification information. The researchers said this allows each pixel imaged to carry “a wealth of data about the biomolecules present.”

This image of tumor and normal brain tissue from a mouse has been colored to show cell nuclei in blue, lipids in red and red blood cells in green. The image shows an area about 200 µm across.

The new instrument achieves enhanced signals by using excitation light quickly and more efficiently, whereas conventional coherent Raman instruments must tune two separate laser frequencies to excite and read different Raman vibration modes in the sample.

“There are a number of firsts in this [study] for Raman spectroscopy,” said Dr. Charles Camp Jr., an electrical engineer at NIST. “Among other things we show detailed images of collagen and elastin — not normally identified with coherent Raman techniques — and multiple peaks attributed to different bonds and states of nucleotides that show the presence of DNA or RNA.”

The BCARS instrument is fast and accurate enough for creation of high-resolution images of biological specimens that contain detailed spatial information on specific biomolecules.

“We’ve engineered a very efficient way of generating our signal with limited amounts of light,” said NIST researcher and chemist Dr. Marcus Cicerone, noting that too much light will destroy the cells. “We’ve been more efficient, but also more efficient where it counts, in the fingerprint region.”

The research was published in Nature Photonics (doi: 10.1038/nphoton.2014.145).

For more information, visit www.nist.gov.