The next time you’re gazing at the “Mona Lisa” at the Louvre in Paris, consider that beneath the subject’s exquisitely enigmatic smile, a new secret has been uncovered about the Leonardo de Vinci masterpiece.

Research published in the Journal of the American Chemical Society suggests that the famously curious, learned, and inventive Italian Renaissance master may have been in a particularly experimental mood when he set to work on the painting early in the 16th century, using a suspected by never-proven technique.

Using X-rays to peer into the chemical structure of a tiny speck of the celebrated work of art, a team of scientists and art historians in France and Britain concluded that the oil-paint recipe that Leonardo used as his base layer to prepare the panel of poplar wood appears to have been different for the “Mona Lisa,” with its own distinctive chemical signature.

“He was someone who loved to experiment, and each of his paintings is completely different technically,” said Victor Gonzalez, the study’s lead author and a chemist at France’s top research body, the CNRS. Gonzalez has studied the chemical compositions of dozens of works by Leonardo, Rembrandt, and other artists.

“In this case, it’s interesting to see that indeed there is a specific technique for the ground layer of ‘Mona Lisa,’” he said.

Specifically, the researchers found a rare compound, plumbonacrite, in Leonardo’s first layer of paint. The discovery, Gonzalez said, confirmed for the first time what art historians had previously only hypothesized: that Leonardo most likely used lead oxide powder to thicken and help dry his paint as he began working on the portrait that now stares out from behind protective glass in the Louvre.

Carmen Bambach, a specialist in Italian art and curator at New York’s Metropolitan Museum of Art, who was not involved in the study, called the research “very exciting” and said any scientifically proven new insights into Leonardo’s painting techniques are “extremely important news for the art world and our larger global society.”

Finding plumbonacrite in the “Mona Lisa” attests “to Leonardo’s spirit of passionate and constant experimentation as a painter – it is what renders him timeless and modern,” Bambach said.

The paint fragment from the base layer of the “Mona Lisa” that was analyzed was barely visible to the naked eye, no larger than the diameter of a human hair, and came from the top right-hand edge of the painting.

The scientists peered into its atomic structure using X-rays in a synchrotron, a large machine that accelerates particles to almost the speed of light. That allowed them to unravel the speck’s chemical make-up. Plumbonacrite is a byproduct of lead oxide, allowing the researchers to say with more certainty that Leonardo likely used the powder in his paint recipe.

“Plumbonacrite is really a fingerprint of his recipe,” Gonzalez said. “It’s the first time we can actually chemically confirm it.”

After Leonardo, Dutch master Rembrandt may have used a similar recipe when he was painting in the 17th century; Gonzalez and other researchers have previously found plumbonacrite in his work, too.