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In vivo imaging: Raman-style
Jeffrey M. Perkel, Ph.D.
BioTechniques, Vol. 54, No. 3, March 2013, pp. 119–121
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The patient lies unconscious on the operating table, brain exposed through a hole in the skull. The tumor – a spherical glioblastoma perhaps 6 mm in diameter – is nothing more than a slightly yellowish opacity on an otherwise pale-pink background.

The surgeon excises the tumor in quarters. He pauses after each step to track his progress using a custom-built imaging system that renders the tumor bright fuchsia. Once the first quadrant is cut away, the tumor takes on the appearance of a pink Pac-Man in mid-chomp. Removing another piece leaves half a pie. Soon, only a sliver of pink remains, and then nothing.

To the naked eye, the operation appears successful. But the surgeon's imager can see what his eyes cannot, rogue cancer cells that have wandered away from the bulk of the tumor to infiltrate the surrounding tissue in the mouse's brain.

Fortunately, the patient was a mouse, and the surgical suite was in a biomedical research lab. Yet the case illustrates one of the biggest patient fears when it comes to tumor removal – leaving a small, unseen piece of cancer behind. Little can allay that fear at the moment. But new imaging techniques based on the so-called Raman effect could eventually alter the emotional calculus. The question is, will they live up to their potential?

A case for Raman

To understand the Raman effect, consider for a moment the physical properties of light that make it possible to read this issue of BioTechniques.

To read a magazine, watch a movie, or marvel at the beauty of your partner's face, light from the object being viewed must pass into your eyes. If that object is a light source, it can be viewed directly. Otherwise, the light that reaches your eyes is reflected off of the object's surface.

In general, when light bounces off an object, the reflected light is of the same wavelength as the incident light, and the reflection is said to be “elastic.” Physicists describe this behavior as the Rayleigh effect, and it explains why things around us appear to have different colors: If you shine a white light on a red billiard ball, most of the light will be absorbed, except the red fraction of the incident light, which bounces toward you. Thus, you see a red billiard ball.

In 1928 physicist Chandrasekhara Venkata Raman, working at Calcutta University, observed that photons can bounce off an object with different energies (and therefore different wavelengths) than the incident light - a discovery that won Raman the 1930 Nobel Prize in Physics.

“Inelastic” Raman reflections are exceedingly rare, amounting to perhaps one in every million reflected photons. Yet each material has a characteristic Raman signature, a fact that is exploited today in chemical and physical science labs that use the effect to probe chemical bonds and the molecular composition of materials. The Raman effect also is increasingly being harnessed for in vitro analyses in biological labs and occasionally, for direct in vivo imaging.

Most in vivo imaging, though, is done using either the medical modalities, such as x-ray or MRI, or more popularly, fluorescence or bioluminescence. Yet neither optical modality is optimal. For one thing, tissues tend to absorb and/or scatter visible light, leading to a diminished signal — a problem compounded by tissue autofluorescence. Tissues are also largely transparent to near-infrared (NIR) wavelengths, an effect you can see for yourself by shining a flashlight behind your hand and watching the color of light that passes through. However, there are relatively few dyes available in this region of the spectrum, making multiplexing tricky. Plus, fluorescent dyes are notoriously unstable, often “bleaching out” long before an experiment is complete.

“There is no one Swiss army knife of imaging that can do everything,” says Sanjiv Sam Gambhir, the Virginia and D.K. Ludwig Professor of Cancer Research, Chair of Radiology, and Director of the Molecular Imaging Program at Stanford University. Each modality has its strengths and weaknesses. The strengths of Raman imaging are sensitivity and high multiplexing levels. Raman signals are sharper than fluorescent spectra, meaning they can be multiplexed more easily. They also don't bleach out, allowing for repeated and long-term imaging and can be both excited and detected in the NIR range.

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