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Studying inner workings of cancer cells in 3-D

BioPhotonics
Feb 2008
Hank Hogan

Doctors would like to achieve early cancer detection in a clinical setting without resorting to contrast agents or invasive techniques. Now researchers at Duke University in Durham, N.C., and at the University of Wisconsin-Madison have demonstrated noninvasive photonics-based techniques that could someday be used for just that.

They used multiphoton microscopy and near-infrared excitation to image cancer in vivo. Duke associate professor of biomedical engineering Nirmala Ramanujam said that the microscopy technique let them access organs of interest without surgery or needles. “Multiphoton microscopy rejects the light from out-of-focus planes, and the NIR excitation of multiphoton microscopy is in the optical window where light penetrates deepest in tissue,” she said.

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Three-dimensional multiphoton image stacks (normal tissue, left; precancerous lesion, right) of the fluorescence lifetime of NADH were examined in vivo in a hamster cheek pouch. The color scale goes from blue for a low NADH fluorescence lifetime to red for a high lifetime. The difference in color between the stacks is from the decrease in the NADH lifetime in precancerous tissue. In the future, images such as these could be used to detect cancers in their earliest stages, reducing morbidity and mortality. Courtesy of Melissa C. Skala, Duke University, and Kevin W. Eliceiri, University of Wisconsin-Madison.

The researchers examined layers of tissue in the cheek pouches of hamsters in three dimensions and with cellular resolution, looking for metabolic markers of cancer. Even in the earliest stages, cancerous cells have higher metabolic demands than normal cells because of their rapid cell division. Thus, the researchers theorized that abnormal cells would have different optical characteristics.

One optical indicator that has been known for years is the ratio of the fluorescence intensity of the metabolic enzymes FAD and NADH — the primary electron acceptor and donor, respectively, in an oxidative phosphorylation process. This redox ratio is sensitive to changes in metabolic rate and vascular oxygen supply. A decrease in the ratio indicates increased metabolic activity, and ex vivo studies have found a similar decrease in cancer cells. The researchers believe that they are the first to look for redox ratio changes in living tissue.

In addition to this standard technique, they examined the fluorescence lifetime of FAD and NADH. Both exist in free and bound states, each with fluorescence lifetime components dependent upon what they are bound to and upon their environment. As a final cancer detector, the researchers looked at cellular morphology.

For all multiphoton imaging, they used an inverted Nikon microscope with a Spectra-Physics Ti:sapphire laser for excitation. They captured fluorescence intensity and lifetime data with a Hamamatsu photomultiplier tube, employing Becker & Hickl electronics for time-correlated single-photon counting. They collected data on animals using excitation first at 800 nm for NADH and then at 890 nm for FAD.

Their studies showed that fluorescence lifetime imaging was the technique that provided the most reliable early cancer indicator, a result that Ramanujam said was surprising but understandable. “The fluorescence lifetimes performed so well because they are robust against the many sources of error that make fluorescence intensities difficult to quantify across multiple animals on multiple days.”

As for the future, one of the group’s aims is to develop a fiber-based optical detection scheme that could be used in a clinical setting. Another is to continue microscopy studies to better understand the fundamental biology underlying the changes in fluorescence lifetime. The two sets of studies — microscopy- and fiber-based — would feed back into one another and proceed iteratively, Ramanujam noted.

PNAS, Dec. 4, 2007, pp. 19494-19499.


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