The Advanced Microscopies team at the Laboratory for Optics and Biosciences has shown that third harmonic generation (THG) microscopy, mainly used to visualize tissue structure, can also help distinguish certain biological pigments. By exploiting their response to different wavelengths, the researchers open new possibilities for label-free multiphoton imaging that can provide complementary information about sample composition.

Multiphoton microscopy is widely used to explore biological tissues in three dimensions. While many approaches rely on fluorescent labels to precisely identify specific structures or molecules, label-free techniques can also make use of the intrinsic properties of biological samples.

Among them, third harmonic generation (THG) microscopy can reveal interfaces and variations in optical properties within tissues. It is therefore particularly useful for providing information about tissue organization and morphology without requiring staining or fluorescent labeling. Until now, however, THG has mainly been used for structural imaging.

In this study¹, Stella Dees et al. investigated another property of THG: its signal can be strongly enhanced in the presence of certain absorbing molecules when they are illuminated at specific wavelengths.

The researchers studied three representative biological pigments: hemoglobin in red blood cells, pteridines found in xanthophores (pigment cells in zebrafish) and melanin in human hair. For each of them, they measured how the THG signal changed depending on the excitation wavelength.

They observed signal enhancements ranging from 10- to 100-fold in these pigmented structures. Importantly, this enhancement did not occur in the same way for all pigments. Each showed a different response to the excitation wavelengths, providing information that can be used to distinguish them.

Distinguishing structures in a living sample

The team then applied this principle to the imaging of live zebrafish larvae. The researchers used an approach called third-order sum-frequency generation (TSFG), which provides simultaneous access to several spectral responses.

By taking advantage of the differences between the pigments, they were able to distinguish red blood cells containing hemoglobin from xanthophores and from surrounding structures.

This simultaneous readout is also particularly useful for imaging living and dynamic samples, as it provides spectroscopic contrast without requiring the excitation wavelength to be changed successively to acquire multiple images.

Going beyond morphological information

These results show that THG could provide more than structural information alone. By exploiting the spectral properties of pigments naturally present in biological samples, THG and TSFG could add a degree of specificity to label-free multiphoton imaging.

This approach is not intended to replace fluorescence imaging, which remains particularly powerful for specifically targeting molecules or biological structures, but could provide complementary information without requiring additional labels.

The work therefore opens new perspectives for studying pigments and other naturally absorbing structures in biological samples, while also highlighting the need for further characterization of their spectral properties and for a better understanding of how light propagation through tissues influences the measured signals.

¹Stella Dees, Júlia Ferrer Ortas, Pierre Mahou, Willy Supatto, Nicolas Olivier, Emmanuel Beaurepaire; Resonant third harmonic generation in biological pigments. APL Photonics 1 August 2026; 11 (8): 086116. https://doi.org/10.1063/5.0341777