<b>Fluorophores in fossil plants: investigation through two photon excitation fluorescence microspectroscopy</b>

Fluorescence emission is a common occurrence in plants and serves multiple functions, such as aiding photosynthesis, attracting pollinators and seed-dispersing mammals, warning insects of chemical defenses, and providing UV protection. Plant fluorescence can serve as an indicator of various physiological processes and provide valuable information about their health and vitality. While fluorescence microscopy has been widely used to study living plants, its application in quantifying the fluorescence of fossilized plants has been limited. Investigating the compounds responsible for fluorescence in fossil plants, whether from original fluorophores or formed during fossilization, can offer valuable insights into fluorescence in ancient plants and the fossilization processes. In this work, we utilized two-photon fluorescence microspectroscopy to spatially and spectrally resolve the fluorescence emitted by different fossil plants, including amber-embedded plants, leaf compression fossils, and silicified wood. The advanced micro-spectroscope utilized in this study, with its pixel-level spectral resolution and line-scan excitation capabilities, allowed us to collect comprehensive excitation and emission spectra with high sensitivity and minimal laser damage to the specimens. By applying linear spectral unmixing to the spectrally resolved fluorescence images, we could differentiate between (a) the matrix and (b) the original biological material or chemical products formed during fossilization. Our analysis suggests that the latter correspond to the durable tissues of the fossils, such as lignin and cellulose. Additionally, we observed potential signals from chlorophyll derivatives, although minerals could have contributed to this fingerprint. Combining this information with chemical analyses of the fossils will improve our understanding of the organic composition of fluorescent plant fossils and enhance the interpretation of fluorescence emission spectra. This research opens doors to exploring the ancient ecosystems and potentially understanding the ecological roles of fluorescence in plants throughout geological time. The findings may have broader implications in the field of paleobotany, offering insights into the evolution of fluorescence in plants and its relevance to ancient plant-animal interactions. Furthermore, the protocols developed as part of this work can be applied to analyze non-plant fossils and biological specimens as well.