Figure 1.
Raman maps of carotenoid pigments in E. blumi.
(A) Bright field micrograph and (B,C) intensity Raman maps of a living E. blumi; Raman map C covers the anterior region of the tardigrade (white rectangle in map B). (D) Bright field micrograph and (E) intensity Raman map of an exuvium containing three eggs of E. blumi. (F) Average Raman spectra (black) and intensity standard deviation (grey) of Raman maps B and E; spectral intensity was re-scaled for better comparison. All Raman maps depict the relative concentration of carotenoid species, measured as the integrated Raman intensity between 1501 cm−1 and 1541 cm−1, corresponding to the intense ν1 C = C stretching vibration band of carotenoids. In C, white arrows indicate spots with high carotenoid concentration corresponding to the eye-spots. The color scale bar on the right of each Raman map has units of total photon counts/Δcm−1 for the Raman shift interval considered.
Figure 2.
Variety of spectral characteristics for carotenoids within a E. blumi specimen.
(A) Image depicting the ν1/ν2 band intensity ratio (“IR”, color scale bar on the right) for the Raman map shown in Fig. 2B. (B) Average normalized intensity of the two subsets of spectra having the lowest and the highest ν1/ν2 ratio, in blue (IR<1.2, 26 spectra) and red (IR>1.45, 21 spectra), respectively; for each subset, the intensity of standard deviation is reported in grey. (B’) Inset with detail of ν1 bands.
Figure 3.
(A) Photoluminescence emission spectrum from the gut of a living E. blumi (excitation at 514.5 nm). Raman bands due to carotenoids (dotted box) are observed together with the chlorophyll fluorescence emission bands at 670 and 737 nm; (A’) inset with fluorescence intensity map (emission at 670 nm); black bar is 50 µm, grey scale bar on the bottom of the Raman map has units of counts at 670 nm. (B) Bottom spectrum: average normalized spectrum (black) and intensity standard deviation (grey) of a set of 300 Raman spectra collected from the moss leaves of G. orbicularis; top spectrum: Raman spectrum from tardigrade’s gut (dotted box in A).
Figure 4.
Effect of induced oxidative stress on the carotenoid content.
(A–B, D–E) Histograms of the integrated Raman intensity in the 1460–1570 cm−1 region from Raman maps of two living E. blumi specimens before any treatment (A, D) and after exposure to 25 mM hydrogen peroxide (B, treated) or water (E, control) for 15 min. For each histogram, the corresponding intensity Raman map depicting the carotenoids distribution (i.e. the intensity at 1521 cm−1) is shown as inset. White scale bars = 200 µm, color scale bars have units of counts. (C, F) Average spectra of the Raman maps before (red) and after (blue) exposure to hydrogen peroxide (maps in A, B) or water (maps in C, D).