Unprocessed microscope images for "Nanoscopic imaging of ancient protein and vasculature offers new insight into soft tissue and biomolecule fossilization"

Description

Raw, unprocessed microscope images for the manuscript: "Nanoscopic imaging of ancient protein and vasculature offers new insight into soft tissue and biomolecule fossilization". These unprocessed images depict type-I collagen fibrils, vascular tissue, and osteocytes of the following bone specimens: extant Bos taurus long-bone, extant Struthio camelus long-bone, extant Alligator mississippiensis long-bone, YG 610.2305 (Rangifer tarandus antler), YG 610.2363 (Bison priscus metatarsal), YG 610.2364 (Equus lambei metatarsal), YG 610.2365 (Bison priscus radius), YG 610.2397 (Mammuthus primigenius innominate), and YG 126.115 (Bison priscus tibia).

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The methods used to collect these curated microscope images are given below (as described by the bioRxiv preprint with doi: 10.1101/2023.12.05.570041): Bone fragments (~200-400 mg) were collected from each specimen and incubated in EDTA (0.5 M, pH 8.0) at room temperature for ~1–5 days, until demineralized organic bone matrix (OBM) was achieved. Care was taken to only sample the interior cortical bone that was the least discolored (in the case of the Pleistocene specimens). For light microscopy of demineralized OBM samples, a Zeiss Axioskop 2 plus microscope was used to image specimens mounted on glass slides, and resultant images were saved as “Tiff” files. For SEM imaging of OBM samples, samples were fixed for 1 h on ice in 2.5% glutaraldehyde with washes in phosphate-buffered saline (PBS) before and after fixation. A graded series of ethanol incubations (1 h at 50%, 1 h 70%, 1 h 95%, 3x 1 h 100% ethanol) was then used to dehydrate the samples. During the series of ethanol incubations, samples were placed within microporous specimen capsules (30 microns, Electron Microscopy Sciences, SKU: 70187-20). For critical point drying, sputter coating, and imaging, samples were transported from the clean lab to the CHANL core facility at the University of North Carolina at Chapel Hill. Samples were critical point dried (Tousimis Autosamdri-931) and sputter coated (Cressington 108 Auto) with ~80 angstroms of palladium-gold metal. Imaging was performed with a Hitachi S-4700 Cold Cathode Field Emission Scanning Electron Microscope with the accelerating voltage set to 15.0 kV. The protocol for light microscopy and SEM imaging of bone vasculature tissue was identical to that used for the OBM, except that a collagenase digestion was performed immediately post demineralization to isolate blood vessels from the OBM. This involved thorough washing of the demineralized OBM (ten times in water purified via a Barnstead E-Pure water purification system) to remove EDTA. The matrix sections were then incubated overnight at 37 °C in a solution of ~1 mg/mL collagenase (collagenase type I, Worthington Biochemical Corporation, 55B7870) dissolved in Dulbecco's PBS (pH 7.2, with 0.1 g/L calcium chloride and 0.1 g/L magnesium chloride added to the standard PBS recipe) with 1% sodium azide (to inhibit potential microbial growth). Isolated vessels were washed in PBS buffer. At this point, either light microscopy or further processing for SEM (starting with glutaraldehyde fixation) was performed, as described for the OBM sections.

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