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Raman spectroscopy and imaging tools for correlative analyses in otoliths sclerochronology

Although tracking the otoliths development as veritable life history capsule for sclerochronology studies appeared increasingly attractive in the past decade [1], the complexity and variability in otoliths biomineral formation in various species is far from being understood. Few studies employed Raman micro-spectroscopy for getting insight into the organic-inorganic fractions and their spatial distribution in otoliths for several fish species. Significant reported drawbacks regarding the otoliths sampling protocol, particularly resins fixation, which can interfere with the original organic matter Raman signal, as well as high variability within individual biomaterial, poor understanding of biomineralization characteristics for various environmental conditions, or lack of correlative data via scanning electron microscopy and energy dispersive X-ray (EDX) spectroscopy, place the sclerochronology approach yet in early stage. Here we present comprehensive Raman micro- spectroscopy and imaging data combined with SEM-EDX line tracking to correlate non- destructively the chemical composition and its high resolution spatial distribution in aragonite otoliths harvested from S. aurata in four regions from Adriatic Sea. Line scan single point Raman spectra have been recorded using a Renishaw InVia Reflex Raman system and a DPSS laser emitting at 532 nm for excitation. Spectra were collected either in short mode, employing just 1 s exposure time, sufficient for inorganic fraction signal, or in extended mode, in the 100-3200 cm-1 range, using 10 s exposure, to complementary assess the organic fraction Raman characteristics attributed to proteins and amino acids. Different collecting optics has been employed to assess the influence of the native anisotropy of otoliths on the Raman signal and imaging data. We show how daily rings are developed with sub-micrometer spatial resolution and further use the information with controlled scan step of the whole sagittal otolith sections. Meaningful common structural characteristics assigned for various otoliths groups as well as variability within individuals are discussed based on the correlated Raman, SEM and EDX data and prospect their linkage with the existing environmental parameters.

Raman spectroscopy, otoliths, sclerochronology, Sparus aurata

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