Fortunately, CARS microscopy proved successful for imaging pheomelanin. "Pheomelanin has a unique chemical structure, there is nothing else like it in the body," Evans said. "So, we started to look at the molecular structure and noticed there was a corresponding unique molecular vibration that might be useful for imaging the pigment with CARS microscopy."

Evans gives much of the credit to his research team, Sam Osseiran and post-doctoral researcher Tracy Wang, for leading the way in developing and refining the CARS microscopy method for imaging pheomelanin. In general, CARS microscopy utilizes two lasers focused on a sample whose energy difference is "tuned" to specific molecular vibrations to generate high resolution imaging information.


"The work led by Tracy was really rather novel application of CARS microscopy to target this biomolecule which no one else has tried to do before," Osseiran said. "We adjusted our system and aligned and tuned everything so that we could specifically target this one melanin pigment, pheomelanin."

Serendipitously, while developing their CARS imaging method, the group found a complementary method that could be used for the simultaneous detection of eumelanin called sum-frequency absorption (SFA) microscopy. SFA makes use of a signal modulation scheme that can detect both species of melanin. This additional imaging tool is important, as most humans produce both species within skin, making mapping the distribution and quantity of both pigments important.

"Sum-frequency absorption imaging allows you to visualize where all the melanin absorbers are within tissue," said Evans. "As both CARS and SFA can be carried out at the same time, these two techniques can be used together to simultaneously image both melanin pigments."

Wang and Osseiran believe their CARS and SFA method could be very helpful for future research on melanoma and its treatment, as well as observing the changes that occur with melanin species in different states. "We are adding another tool to our utility belt here in our investigations of melanoma," Osseiran said.

The study's original motivator, David Fischer, believes that a very important benefit of the work might be its potential role in diagnosing cancer.

"This may offer a brand-new tool for early diagnosis for some of the most lethal melanomas, possibly at a stage when they might still be curable," said Fisher. "Time and time again, it is proven that early diagnosis saves lives."

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