DOI: 10.7910/dvn/itlde7
title: Collagenous sample ToF-SIMS spectra for "Nanoscopic imaging of ancient protein and vasculature offers new insight into soft tissue and biomolecule fossilization"

description: Raw, unprocessed ToF-SIMS spectra corresponding to Figure 2, Figure 3, and Figure 4 of the manuscript: "Nanoscopic imaging of ancient protein and vasculature offers new insight into soft tissue and biomolecule fossilization". These spectra represent raster scans of the surface of type-I collagen fibrils isolated from 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). Spectra are also presented that represent raster scans of the following protein standards: type-I collagen, bovine serum albumin, hemoglobin. Additionally, spectra taken of the blank silicon wafer surface are also presented.
description: The methods used to collect these curated ToF-SIMS spectra are given below (as described by the bioRxiv preprint with doi: 10.1101/2023.12.05.570041):

Small bone fragments (~100 mg) were placed in 0.24 M hydrochloric acid to demineralize. After complete demineralization (~1-2 days), OBM sections were removed and washed with E-Pure water. For vascular tissue analysis, the OBM sections were then incubated overnight at 37 °C in 1 mg/mL collagenase (collagenase type I, Worthington Biochemical Corporation, 55B7870) with 1% sodium azide dissolved in Dulbecco’s PBS. Isolated blood vessels were washed twice with E-Pure water. For the collagen I, BSA, and hemoglobin protein standards, chloroform/methanol extraction was necessary to remove residual lipids, despite the “purified” standards having been purchased from companies. A few grams of each standard was incubated for ~30 minutes with agitation in a 2:1 mixture of chloroform:methanol. The chloroform:methanol solution was then decanted off, and the protein standards washed once with fresh chloroform:methanol solution. The wash solution was decanted off, and the protein standards were left to air dry in a chemical fume hood. Immediately (a few hours before) prior to specimen mounting, silicon wafers (provided courtesy of the Analytical Instrumentation Facility at NC State University) were subjected to an RCA-1 cleaning protocol. The RCA-1 cleaning solution consisted of a heated 5:1:1 mixture of 5 parts E-Pure H2O, 1 part 25% NH4OH, and 1 part 30% H2O2. Cleaned wafers were washed twice with E-Pure water and left to dry in a laminar flow hood.

For specimen mounting, OBM and blood vessel samples were suspended in sterile E-Pure water and pipetted directly onto the RCA-1 cleaned silicon wafers and allowed to air dry in a laminar flow hood. Protein standards were mounted directly onto copper conducting tape, provided courtesy of the CHANL core facility at UNC Chapel Hill. The specimens were then immediately transported to the Analytical Instrumentation Facility at North Carolina State University and placed under vacuum within the ToF-SIMS instrument. Sample analysis took place the following day.

A TOF-SIMS V (ION TOF, Inc.) was used to analyze the prepared OBM, blood vessel, and protein standard samples. Analyses were performed with a Bi3+ liquid metal ion gun at 45o incident to the sample surface under an ultra-high vacuum of ~7.9x10-10 mbar. Regions of 50x50, 100x100, or 150x150 µm2 were scanned using the instrument’s high current-bunched (high mass resolution) setting with a beam diameter ~10 microns, a pulse width of ~1 ns, and a cycle time of 100 microseconds. Data was recorded in the instrument’s positive mode for OBM sections and protein standards, and in the negative mode for vascular tissue. Images obtained were 128x128 pixels with 1 shot/pixel at an approximate target current of 0.33 pA (this was checked using a Faraday cup prior to each analysis session to ensure the static “limit” was not exceeded). Primary ion dosage was kept below 1012 ions/cm2 to ensure all analyses were performed under static conditions. Resultant spectra had a mass resolution of ~3,000-6,000 m/Δm, dependent on degree of charging and analysis area topography. An electron flood gun was used for charge compensation to mitigate charging-related issues such as degraded mass resolution and attenuated secondary ion signal. When degraded mass resolution (or even peak splitting) due to sample topography were encountered, relatively flat regions of interest within the analysis area were selected to mitigate this issue.