Pushing Raman spectroscopy over the edge: purported signatures of organic molecules in fossil animals are instrumental artefacts

Abstract

Widespread preservation of fossilized biomolecules in many fossil animals has recently been reported in six studies, based on Raman microspectroscopy. Here, we show that the putative Raman signatures of organic compounds in these fossils are actually instrumental artefacts resulting from intense background luminescence. Raman spectroscopy is based on the detection of photons scattered inelastically by matter upon its interaction with a laser beam. For many natural materials, this interaction also generates a luminescence signal that is often orders of magnitude more intense than the light produced by Raman scattering. Such luminescence, coupled with the transmission properties of the spectrometer, induced quasi-periodic ripples in the measured spectra that have been incorrectly interpreted as Raman signatures of organic molecules. Although several analytical strategies have been developed to overcome this common issue, Raman microspectroscopy as used in the studies questioned here cannot be used to identify fossil biomolecules.

Validation of biosignatures confirms the informative nature of fossil organic Raman spectra

“…Alleon et al (21) presented different observations and arguments in support of their assertion that the disputed spectra are artefactual in nature. Their arguments relied primarily on a frequency contribution analysis which, as far as we can determine, has not been applied by others to baselined Raman spectra.…”

“…Alleon et al (21) downloaded a freely available RazorEdge filter transmission spectrum (21, fig. 2a), which resembles a periodic, sinusoidal wave function, from the commercial website 'Semrock' (www.semrock.com/FilterDetails.aspx?id=LP03-532RE-25).…”

“…2b, continuous red band). Periodic wavelet transform analysis (21), which is commonly applied to acoustic or precipitation data (reviewed in 22), is not usually applied to Raman spectra beyond determining the ideal baselines for raw data (23), and is inferior to Principal Component Analyses of spectral data for assessing Raman spectral signatures (16,24). We are unaware of examples in the literature where wavelet transform analysis has been applied to spectra that were already baselined and normalized, which is the nature of the published spectra analyzed by Alleon et al (21).…”

“…This analysis decomposes a given wave form, whether periodic or not, into its frequency components, and displays their contributions to the source wave form by quantifying the extent to which a sinusoidal peak in each frequency contribution needs to be scaled to resemble the original shape of the curve. Alleon et al (21) encoded this information in the form of a heat map (figs. 1a, c, 2b, 3f).…”

“…A diversity of studies conducted by different laboratories (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) have recovered very similar patterns in the molecular makeup of fossil organic matter in independently acquired in situ Raman spectra. However, a recent Perspective by Alleon et al (21) claimed that the results of a selected subset of these studies (3,4,6,11,12,15) are invalid, based on the assertion that published spectra, despite resembling previous independent Raman analyses (7-10, 13, 14), are the result of instrumental artefacts reflecting sinusoidal edge filter ripples. The disputed studies are based on in situ Raman spectra for a diversity of modern, experimentally matured, and fossil tissues of different types, as well as sedimentary host rocks.…”