Fibres and cellular structures preserved in 75-million-year-old dinosaur specimens

Abstract

Exceptionally preserved organic remains are known throughout the vertebrate fossil record, and recently, evidence has emerged that such soft tissue might contain original components. We examined samples from eight Cretaceous dinosaur bones using nano-analytical techniques; the bones are not exceptionally preserved and show no external indication of soft tissue. In one sample, we observe structures consistent with endogenous collagen fibre remains displaying ∼ 67 nm banding, indicating the possible preservation of the original quaternary structure. Using ToF-SIMS, we identify amino-acid fragments typical of collagen fibrils. Furthermore, we observe structures consistent with putative erythrocyte remains that exhibit mass spectra similar to emu whole blood. Using advanced material characterization approaches, we find that these putative biological structures can be well preserved over geological timescales, and their preservation is more common than previously thought. The preservation of protein over geological timescales offers the opportunity to investigate relationships, physiology and behaviour of long extinct animals.

Figure 1. Density-dependent colour scanning electron micrographs of samples of NHMUK R12562, an ungual claw of an indeterminate theropod dinosaur, and NHMUK R4493, ribs from an indeterminate dinosaur.

(a) Amorphous carbon-rich material (red) surrounded by dense material (green). Scale bar, 5 μm. (b) Erythrocyte-like structures composed of carbon surrounded by cement. Scale bar, 1 μm. For comparison, fixed blood from an emu (Dromaius) is shown in Supplementary Fig. 2c, d. Fibrous structures. Scale bar, 5 μm in (c) and 1 μm in (d).

Figure 2. Scanning electron micrographs and 3D reconstructions from serial sections of erythrocyte-like structures and fibrous material.

(a, b) SEM using a backscattering detector showing cross-sections of erythrocyte-like structures c in NHMUK R12562; arrows indicate dense internal material; scale bar, 0.5 μm. (c) 3D reconstruction of serial sections of an agglomeration of erythrocyte-like structures showing: I, upwardly concave external morphology and II, dense structures observed in the interior of the erythrocyte-like structures. (d) SEM using a backscattering detector from section of fossilized bone of NHMUK R4493; arrows indicate less dense zones inside bone matrix; scale bar, 1 μm. (e) 3D reconstruction of the less dense zones in d, showing elongated, fibre-like morphology and alignment. Random colours assigned to individual fibres to differentiate between them.

Figure 3. Scanning transmission electron microscopy (STEM) analysis of NHMUK R4493.

(a) Bright-field STEM micrograph depicting fibre fragments showing a banded pattern consistent with banding typically observed in collagen fibrils. The arrow indicates the fibre analysed in e and f. Scale bar, 200 nm. (b) Dark-field STEM micrograph showing detail of fibres in a. Scale bar, 100 nm. (c) STEM of fibre analysed by electron energy loss spectroscopy (EELS) indicating spectra locations. Scale bar, 50 nm. (d) EELS spectra showing a carbon peak on the fibre. A smaller peak is seen on the adjacent material. (e) Grey intensity distribution over fibre (indicated by the arrow) in a. Vertical lines in red are spaced 67 nm apart and generally correspond with peaks in grey intensity. (f) Periodicity characterization of e confirming the ∼67 nm banding. (g) Diagram representing the structure of a generic collagen molecule that produces 67 nm banding; I banded collagen fibrils surrounded by bone mineral matrix; II individual fibrils are composed of numerous collagen molecules arranged to produce 67 nm banding; III the canonical collagen triple helix.

Figure 4. Sample preparation by focused ion beam (FIB) for mass spectroscopy analyses of NHMUK R4493, NHMUK R12562 and fixed emu blood.

(a) NHMUK R4493 with sampled location. (b) SEM of FIB sample preparation sequence: I, sample surface; II, platinum protecting layer; III, trench milling; IV, sample on copper grid holder ready for mass spectra acquisition. (c) NHMUK R12562 with sampling location. Scale bar, 5 μm. (d) Sample on grid holder before mass spectra acquisition. Scale bar, 5 μm. (e) SEM image of fixed emu blood with sampling location. (f) Sample on grid holder before mass spectra acquisition. Scale bar, 5 μm. The mass spectrum was obtained from the fresh surface in IV (d and f).

Figure 5. Mass spectra detail of NHMUK R4249 (sample with banded fibres), NHMUK R4493 (sample with 67 nm banded fibres) and NHMUK R12562 cement.

(a, d and g) Peaks are associated with glycine fragments. (b, e and h) Peaks are related to alanine fragments. (c, f and i) Peaks are related to proline fragments.

Figure 6. Mass spectra of fixed emu blood, erythrocyte-like structures present in NHMUK R12562 and cement surrounding these erythrocyte-like structures, all prepared by FIB.

(a) Mass spectrum of emu blood with inset detailed region between 460 and 475 m/z. (b) Mass spectrum of erythrocyte-like structures present in NHMUK R12562, with inset detailed region between 460 and 475 m/z. (c) Mass spectrum of cement surrounding the erythrocyte-like structures, with inset detailed region between 460 and 475 m/z. Blue arrows indicate the regions of main peaks in each spectrum that are present only in the mass spectra of fixed emu blood and erythrocyte-like structures. Red arrows indicate the regions of main peaks in each spectrum that are present only in the mass spectra of erythrocyte-like structures and cement surrounding erythrocyte-like structures.