Blood Clots in Dinosaur Bones
under certain conditions, and has had widespread medical use [49–54]. UVFL examination of dinosaur bone is rare with only a few studies of UVFL application to dinosaur specimens including examina- tion of dinosaur fossil feathers [55] and bones of birds related to dinosaurs [57]. However, these did not utilize autofluo- rescence microscopy. UVFL autofluores- cence microscopy (and fluorophore-based microscopy) of dinosaur bone thin sec- tions represents a unique opportunity to study well-preserved tissue and cellular morphology of dST in situ. In order to generate autofluorescence,
Figure 12: Triceratops frill. A steel pin placed on the surface of the field diaphragm is imaged clearly through a fluorescing clot opening demonstrating that neither the embedding polymer nor the ground surface is autofluorescing. Scale bar, 100 μm.
Results All Triceratops ground sections exhibited heavy clotting
in all vessel channels except those that were closest to the bone edge, a processing artifact. Tese mature bones were typical; presenting newly formed Haversian systems as well as older, partially resorbed ones. Irregular interstitial areas were also observed, as were the concentric lamellae encircling canals. Fine radiating canaliculi and lacunae were apparent. Most microvascular channels, particularly those under
200 µm in width, were completely obstructed by clots (Figures 8–10, white arrows). Some had small openings within the clots (Figures 8a, 8b, 11b, asterisks). Tese clear openings were situ- ated near lumen centers in canals. Partial clearing of canals could mean that clots might have been restricted to short lengths in veins, possibly a result of deep vein thrombosis in settings of low shear flow such as at venule-cuspid junctions [46]. Alternately clots may have been dislodged, perforated, or lost during processing, creating openings. Angular crystalline structures, light brown and somewhat translucent in nature (Figures 8a and 11a), are clearly seen in the open spaces within clots. Tese were observed in every vessel canal. Clots exhib- ited a dense, narrow outer marginal layer (black arrows Figures 8b, 9b, 10b, 11b), and clot material never crossed over this mar- gin into compact bone, remaining isolated in canals.
Ultraviolet Fluorescence and Autofluorescence of Bone Autofluorescence is an intrinsic property of some biologi-
cal compounds (especially within cells and tissues) that receive and are excited by energy, such as light energy. When energy is transferred the compounds fluoresce or give off light energy as the influx of excitation energy stabilizes [47,48]. Many biologi- cal molecules and structures such as collagen, elastin, flavins, porphyrins, and pyroxidines autofluoresce in response to UV illumination. Ultraviolet fluorescence microscopy (UVFL) is a useful tool for imaging specific features of bone, which autofluoresces
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high levels of light energy with a broad- based spectrum are directed at specimens being examined. Te emission spectrum of the 100 W mercury burner used in this study provides high irradiance between 275–500 nm [56]. Tis would certainly supply enough excitation energy to elicit
responses from iron along several of its emission lines including 374.55 nm even if bound in clots. Tat line has the highest auto- fluorescence emission intensity for Fe I [58], however 26 other strong emission lines have been reported for Fe I and II between 234.34 nm and 400 nm [58]. Clots imaged here emitted light between 375 and 450 nm, thus emission falls within the expected range of autofluorescence wavelengths for Fe I and Fe II. It could be argued that the brightly reflective layer is just
an artifact of grinding during processing. To test this possi- bility, the point of a pin at 100×, placed just at the sub-stage field diaphragm of the microscope, using low intensity trans- mitted light, was optically resolved while focusing through an opening in the center of an autofluorescing clot (Figure 12). If the embedding polymer was autofluorescing, or if a grinding artifact might be creating a roughened surface that might oth- erwise be autofluorescing, the point of the pin (13 cm below the specimen) would be obscured or unresolved. Moreover, the clot completely obscures the pin shaſt in Figure 12, yet the pin shaſt is visible through the bone mineral, thus the clot is denser than the bone itself.
Discussion and Conclusions Tis report of blood clots in dinosaur bones is contro-
versial, much as reports of other blood related products has been [4–7]. Nevertheless, although tentative, observations presented here warrant further investigation into the nature of dinosaur blood clots, especially the concentrations and oxidation state of iron in them. Iron response to UV light is significant and shows up as a brightly reflective bluish-white layer with embedded angular objects (Figures 8b, 9b, 10b, 11b, 12). We conclude that the non-fluorescing angular objects are crystallized blood products within each vessel occlusion. It is unlikely that the brightly reflective layer and the angular crystals within them represent an unconsolidated influx of soil matrix deep into frill and horn bone specimens because the emission from the brightly reflective layer is uniform over all sections studied and is only disturbed by embedded
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