SEM analysis

We examined fossilized bones samples using SEM from eight dinosaur specimens (Supplementary Table 1) that show no external indication of soft tissue. Density-dependent colour SEM21 obtained from the surface of minute pieces broken off from samples with tweezers revealed numerous low-density structures suggestive of soft tissue in sample NHMUK (Natural History Museum, London, UK) R12562, an ungual claw of an indeterminate theropod dinosaur (Fig. 1 and Supplementary Figs 1,3,4). Among these structures ovoid structures highly reminiscent of avian erythrocytes22,23 (Fig. 1a,b) were imaged and ranged in length from 1.2 to 3.2 μm (average 1.8±0.4, n=35). In addition to the erythocyte-like structures, in four other specimens the SEM analysis also showed fibrous structures similar to calcified collagen fibres found in modern bone24,25,26 (Fig. 1c,d, Supplementary Table 1 and Supplementary Fig. 5).

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). Full size image

Elemental analysis using energy dispersive X-ray spectroscopy (EDS) established that all these structures are enriched in carbon, in contrast with the surrounding denser tissue/cement, which is composed of carbon, oxygen, iron, silicon and aluminum (Supplementary Fig. 6). Martill and Unwin27 suggested that iron might be expected when looking for blood in fossils, and in a recent study, Greenwalt et al.15 found haemoglobin-derived porphyrin in the abdomen of a blood-engorged Eocene mosquito. Analysis of the material from the mosquito abdomen suggested it is 9% iron by weight. Given that red blood cells ordinarily contain only around 0.3% iron by weight, Greenwalt et al. suggested the unusual high percentage of iron measured may be due to a taphonomic concentration15. Our EDS analysis of the erythrocyte-like structures in sample NHMUK R12562 shows that iron is present in higher quantities in areas of the fossilized bone where no erythrocyte-like structures are preserved. This is to be expected because an iron concentration of only 0.3% by weight typical of a red blood cell is below the detection limit of EDS28. To confirm this hypothesis, we carried out EDS on emu blood and also did not detect iron, thus confirming the detection limit of the technique (Supplementary Fig. 7).

Internal structures investigated by FIB

To evaluate the internal structure of the features imaged by SEM, we initially used a FIB to section the erythrocyte-like structures from NHMUK R12562 and fibrous structures present in NHMUK R4493, rib fragments from an indeterminate dinosaur (Supplementary Fig. 1). Interestingly, denser structures (as imaged by the backscattering detector) were observed inside the erythrocyte-like structures (Figs 2a,b). To further investigate these features, three-dimensional (3D) reconstruction of serial sections of an agglomeration of erythrocyte-like structures (Fig. 2c and Supplementary Movie 1) showed that each was concave, whereas the dense internal features resembled nuclei and could be clearly distinguished within each of them.

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. Full size image

The serial sections from NHMUK R4493 showed less dense linear zones surrounded by a denser matrix within the samples (Fig. 2d). 3D reconstruction of the less dense zones (Fig. 2e and Supplementary Movie 2) clearly showed that these are 3D fibres with an average diameter of 54±9 nm (n=30) and are preferentially aligned in a specific direction (Supplementary Fig. 8).

TEM analysis

To evaluate the ultrastructure of these fibres, TEM was carried out on sections from NHMUK R4493, NHMUK R4243, an astragalus of a hadrosaurid, NHMUK R4249, an ungual phalanx of a hadrosaurid and NHMUK R4864, a hadrosaurid tibia (as a control, rabbit bone (Supplementary Fig. 9) was also prepared using the same method). All samples were prepared by FIB, a method that would preserve the mineral and organic material21. The sections were obtained from the interior of each sample, ruling out modern surface contamination. Unexpectedly, from three of the samples (NHMUK R4493, NHMUK R4249 and NHMUK R4243) TEM micrographs showed obvious fibrous structures (Fig. 3a,b,c and Supplementary Fig. 10) containing carbon (Fig. 3c,d). One sample (NHMUK R4493) also showed, for the first time in a dinosaur bone, a clear ∼67 nm banding, that is typical of the banding observed in collagen (Fig. 3e), for the length of the preserved fibre.

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. Full size image

In modern day bone collagen, the ∼67 nm banding is considered a diagnostic ultrastructure characteristic and arises from the arrangement of fibrils to form the quaternary structure of the collagen protein29,30,31,32,33,34,35,36,37,38,39,40,41 (Fig. 3f). Although 67 nm banding can be considered, in some cases, a finger print of specific proteins, this method is not able to show the chemical composition of the structures imaged. If collagen is completely degraded, this banding is no longer seen, due to loss of the quaternary structure of the protein. Therefore, the observation of a ∼67-nm banding in the fibrous structures of fossilized samples here is very exciting, as it is consistent with a preservation of the ultrastructure of putative collagen fibres over a time period of 75 million years. Before this finding, the oldest undegraded collagen recorded (based on mass spectrometry sequencing and peptide fingerprinting) was about 4 million years old20.

Mass spectrometry analysis

Finally, a thick section (∼20 × 15 × 5 μm3) of samples was extracted using FIB (see Fig. 4 for details of sample preparation) and analysed with Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) to probe for the presence of amino acids. Sections were obtained from an agglomeration of erythrocyte-like structures and cement surrounding these from specimen NHMUK R12562, fixed emu blood, three fossils showing calcified fibres (NHMUK R4493, NHMUK R4249, NHMUK R4864), rabbit bone and a fossil not presenting sign of calcified fibres (NHMUK R12562). As a control, a mass spectrum from the copper grid holding the samples was also obtained.

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). Full size image

Extraction of a fresh surface for ToF-SIMS analysis by the FIB placed inside the electron microscope guarantees correct localization of sampling and provides a clean, smooth surface for analysis that eliminates topographical artefacts common to ToF-SIMS measurements42. Moreover, in the case of the cement from specimen NHMUK R12562 and NHMUK R4493 (where the bone was directly sampled), this method rules out the possibility of modern contamination, as the surface exposed is inaccessible to any contaminant.

The positive mass spectrum obtained from NHMUK R4493 showed peaks corresponding to fragments of the amino acids glycine, alanine, proline and others14,43,44 (Fig. 5 and Supplementary Fig. 11; for attribution of peaks see Supplementary Table 2). Moreover, several peaks present in the spectrum from rabbit bone and the fossil samples showing calcified fibres are not present in the results for the fossil without calcified fibres or on the negative control from the copper grid holding the samples (Supplementary Figs 12 and 13). Detection of fragments of the amino acids normally found in collagen supports the results obtained from TEM analysis where the ∼67 nm banding is consistent with potential preservation of the original quaternary structure of the protein.

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. Full size image

The positive mass spectra obtained from the erythrocyte-like structures of NHMUK R12562 were compared with spectra obtained from emu blood fixed in paraformaldehyde and with the cement surrounding the structures (Fig. 6). The spectra obtained from four different regions of the dinosaur bone containing erythrocyte-like structures are surprisingly similar to the spectra obtained from emu blood.

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. Full size image

As expected, spectra obtained from emu blood (Fig. 6a) indicate the presence of several components, as the spectra were obtained from sections that comprised whole blood. Some of the main peaks could be tentatively assigned to components present in whole blood such as: 318 m/z to folic acid45; 462 m/z to hydroxycholesterol46,47 and the peak at 572 m/z to ceramide (usually present in cell membranes48). The same pattern of peaks can be seen from the spectra obtained from the erythrocyte-like structures (Fig. 6b) and this is clearer when a higher magnification is applied to those spectra (Fig. 6a,b inset). Moreover, it is clear that the mass spectrum from the cement surrounding erythrocyte-like structures (Fig. 6c) is different from both the spectrum from erythrocyte-like structures and that from emu blood. It is particularly interesting that the mass spectrum from the erythrocyte-like structures has some features of both the mass spectra from emu blood and cement (Fig. 6b).

The presence of several protein residues preserved in the erythrocyte-like structures of NHMUK R12562 could explain differences between the spectra obtained by us and previous spectra obtained from fossilized blood samples such as that obtained by Greenwalt et al.15, where only haemoglobin-derived porphyrin was preserved in the fossil record. Finally, it is clear that the spectrum from cement is different from the spectra obtained from emu blood and from erythrocyte-like structures. Similarities between cement and the erythrocyte-like structures are expected because the cement is also present in the sample of the erythrocyte-like structures and fragments of the putative proteins are likely present in the cement, even if they cannot be visualized by electron microscopy. The mass spectra obtained from emu whole blood are slightly different in comparison with those of the erythrocyte-like structures, which is to be expected because of the presence of organic components derived from additional blood proteins that have not degraded in the emu sample, but are absent in the erythrocyte-like structures, and also because the emu blood was fixed with paraformaldehyde.

Partial least square—discriminant analysis (PLS-DA) of mass spectra from four dinosaur regions presenting the erythrocyte-like structures samples and four emu samples, collected from different regions and from a cement sample was carried out. The cross-validated PLS-DA were carried out in the region between 300 and 700 m/z (stressing, any organic differences between the components of the samples (Supplementary Figs 14 and 15)). The spectra from emu blood samples and those from areas of NHMUK R12562 containing erythrocyte-like structures are enclosed within the 95% confidence ellipse and, as a group, may be compared with the cement sample, which falls outside the ellipse. This is another indication that the biochemical fingerprints of the spectra of emu blood samples and of areas of NHMUK R12562 containing erythrocyte-like structures are very much alike.

Within the dinosaur samples on average, the erythrocyte-like structures are ∼2 μm in length. This is somewhat smaller than erythrocytes of birds, which range from 9 to 15 μm in length22,49; emu blood cells in our sample were 9±2 μm (n=17). The structures consistent with putative erythrocytes in the fossil could well have been deformed and it is quite probable that these structures have undergone some shrinkage during fossilization.

Red blood cell size is known to correlate with metabolic rate in many vertebrate clades, including reptiles and birds49. Discoveries of red blood cells in a range of dinosaur taxa offer a potential independent method of assessing relative metabolic rates and may help to ascertain when and how the transition from ectothermy to endothermy took place on the avian stem lineage.

The potential for future research into the metabolic rate of extinct animals based on erythrocytes is promising because in this study, putative soft tissue (either erythrocyte-like structures, collagen-like, fibrous structures or amorphous carbon-rich structures (Supplementary Fig. 7)) was observed in six of our eight dinosaur specimens (Supplementary Table 1). Incredibly, none of the samples showed external indicators of exceptional preservation and this strongly suggests that the preservation of soft tissues and even proteins is a more common phenomenon than previously accepted.

These results show that to determine the presence of soft tissue in fossils a new synergistic approach needs to be applied where micro/nano-analytical methods are utilized to their full potential.

The common preservation of soft tissues could pave the way for cellular investigations of extinct animals, shedding light on aspects of physiology and behaviour that have been previously inaccessible to palaeontologists and inaugurating a new and exciting way to do paleontology.