A label-free imaging technique can distinguish biological pigments

Label-free multiphoton imaging provides noninvasive, 3D visualizations of intact biological tissues. In particular, third harmonic generation (THG) microscopy — which scans a pulsed laser beam across a sample to find changes in refractive index — can help characterize these samples by identifying micron-scale interfaces and heterogeneities.

But THG microscopy lacks chemically-specific information for use in certain biomedical applications. Following up on previous studies of THG responses in hemoglobin, Dees et al. imaged red blood cells and yellow pigmented cells called xanthophores in zebrafish larvae, along with melanin in human hair, to identify whether THG could distinguish between different pigments in a sample.

“Since xanthophores and red blood cells are present in the same regions of zebrafish larvae, it was a natural idea to explore both pigments in the same samples,” said author Emmanuel Beaurepaire. “Melanin is also a very important biological pigment and was an interesting target for analyzing the generality of the THG resonance mechanism in pigments.”

They found THG could identify spectral signatures in all three sample types, confirming the method is sensitive to different types of pigments within live biological samples. In the xanthophores, the THG spectrum was shifted towards ultraviolet wavelengths, and in hair, the THG signal levels depended on melanin concentration.

“All of these pigmented structures exhibited resonantly-enhanced THG,” Beaurepaire said. “This suggests the spectroscopic THG could have several interesting applications in the life sciences.”

The group says that further optimizing the technique will allow it to work alongside other imaging methods to acquire complementary information about a biological sample within a single noninvasive measurement.

Source: “Resonant third harmonic generation in biological pigments,” by Stella Dees, Júlia Ferrer Ortas, Pierre Mahou, Willy Supatto, Nicolas Olivier, and Emmanuel Beaurepaire, APL Photonics (2026). The article can be accessed at https://doi.org/10.1063/5.0341777 .