Seeking carotenoid pigments in amber-preserved fossil feathers
Animal colours can be richly informative about aspects of behaviour such as foraging ecology and mate preference. Birds in particular display many striking hues and complex patterns of pigmentation. Examples of plumage adaptations include brightly coloured feathers for enticing potential mates, as well as cryptic patterns that allow a bird to hide in plain sight1,2,3. By analogy with modern birds, the behaviours and habitats of ancient birds and other feathered dinosaurs may be inferred from pigments in fossil plumage. Methods for describing one class of plumage pigment (melanins) have recently been developed for ancient feathers4,5. Modern birds display six chemically-distinct classes of feather pigment, of which carotenoids are the most common after melanins; carotenoids mostly confer yellow, orange and red colours to feathers6,7. The two key challenges for describing carotenoids in ancient plumage are 1) to find an environment that preserves molecular evidence for carotenoids, and 2) to ascertain a technique that provides unequivocal evidence for carotenoids. We focused on feathers in amber because encapsulation within amber may insulate carotenoid molecules against diagenetic alteration, and such preserved pigments could be detected with Raman spectroscopy. Carotenoids have an unequivocal Raman spectral signal that is greatly enhanced by a resonance effect, allowing trace amounts to be detected8. Furthermore, Raman spectroscopy can be performed without sample preparation or destruction. Edwards and colleagues9 showed that inclusions in amber can be studied with minimal or no interference from the amber matrix when Raman spectra are collected with confocal optics and 1064 nm excitation. With conventional Raman microscopy, laser light is channeled through a microscope objective towards a sample, and then scattered light is channeled back through the same objective towards a detector. Light scatters from the entire sample volume that is penetrated by incident light, and the greatest concentration of scattered photons are typically returned from the focal plane of the incident light. Accordingly, light is scattered from both the surface and internal volume of a translucent sample. Confocal Raman microscopy differs from conventional Raman microscopy by the inclusion of a confocal pinhole above the microscope objective. The pinhole allows only the photons scattered from the focal plane to reach the detector. For a translucent sample with an inclusion (e.g. amber containing a fossil feather), the inclusion can be positioned in the focal plane of the incident light and only the light scattered from the inclusion will reach the detector. Hence, confocal Raman microscopy usefully provides chemical information about an inclusion with minimal or no interference from the surface or surrounding matrix. Near infrared excitation wavelengths are typically less sensitive than visible wavelengths when studying pigments with Raman spectroscopy. However, visible excitation wavelengths tend to induce fluorescence when interacting with amber9. Thus, from a signal vs. noise perspective, a Raman spectrum of an inclusion in amber collected with a near infrared wavelength can be substantially more informative than a Raman spectrum of the same inclusion collected with a visible wavelength. A confocal Raman microscope with a near infrared (NIR) laser is thus an ideal tool for seeking carotenoids preserved in amber. We first conducted a pilot study to determine if the carotenoid pigments of a modern feather could be detected through an amber matrix. A yellow and black wing feather from a greenfinch (Carduelis chloris) was placed underneath a polished piece of amber and analysed with Raman spectroscopy. In a second series of experiments, we analysed six fossil feathers preserved in amber using Raman spectroscopy and light microscopy. Evidence for pigmentation in the fossil feathers was sought with Raman spectroscopy, and the preservation of fine-scale morphology was studied with light microscopy. Finally, we collected Raman spectra and scanning electron microscopy images from an ancient feather preserved as a carbonised compression fossil. Compression fossils have previously provided evidence for melanin pigmentation4,5, and here we compared Raman spectral evidence from two types of fossil feather (1: preserved in amber; 2: preserved in lake sediment) with spectra from a modern feather.
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