Towards Raman Spectroscopic Characterization of Tardigrade Molecular Composition

ABSTRACT Tardigrades are microscopic animals known for their sustainability to various stressful conditions. The mechanisms underlying tardigrades adaptation are of great interest, but in vivo study of their internal composition is challenging. Raman spectroscopy is an attractive technique for such studies; however, no successful measurements of nonresonant Raman spectra from major organic molecules were reported for tardigrades. Here, we demonstrate that low‐fluorescence Raman spectra with a high signal‐to‐noise ratio can be measured in living tardigrades immobilized by tun formation. We compare results obtained from Raman experiments with different excitation wavelengths (532 and 780 nm) and assess the phototoxicity effect. It was found that laser radiation at a wavelength of 532 nm and a power of 20 mW is lethal to tardigrades. After irradiation using 780 nm (80 mW), tardigrade survival rate exceeded 70%. In addition to the previously observed contributions of carotenoids and calcium carbonate, we detect the contributions of cytochromes, other proteins, lipids, nucleotides and visualize their spatial distribution. It was discovered that the midgut region of tardigrades may contain crystalline guanine monohydrate with grain sizes of ~3 μm. In this work, we demonstrate the suitability of Raman spectroscopy for the molecular analysis of tardigrades, opening new horizons for research on their stress adaptation.