Preservation of Bone Collagen from the Late Cretaceous Period

Tissue and Cell-Like Structures Preserved in Dinosaur Bone. Mary Higby Schweitzer , Alison E. Moyer , Wenxia Zheng. PLOS ONE 2016 11 (2), e0150238...
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Preservation of Bone Collagen from the Late Cretaceous Period Studied by Immunological Techniques and Atomic Force Microscopy R. Avci,*,† M. H. Schweitzer,‡,§ R. D. Boyd,† J. L. Wittmeyer,‡ F. Tera´n Arce,† and J. O. Calvo| Montana State University, Department of Physics, EPS 264, Bozeman, Montana 59717, North Carolina State University, Department of Marine, Earth and Atmospheric Sciences, Raleigh, North Carolina 27695, North Carolina Museum of Natural Sciences, Raleigh, North Carolina 27601, and Museo de Geologia Palentologia, Universitad Nacional del Comahue, Neuquen, Argentina Received September 16, 2004. In Final Form: February 1, 2005 Late Cretaceous avian bone tissues from Argentina demonstrate exceptional preservation. Skeletal elements are preserved in partial articulation and suspended in three dimensions in a medium-grained sandstone matrix, indicating unusual perimortem taphonomic conditions. Preservation extends to the microstructural and molecular levels. Bone tissues respond to collagenase digestion and histochemical stains. In situ immunohistochemistry localizes binding sites for avian collagen antibodies in fossil tissues. Immunohistochemical studies do not, however, guarantee the preservation of molecular integrity. A protein may retain sufficient antigenicity for antibody binding even though degradation may render it incapable of original function. Therefore, we have applied atomic force microscopy to address the integrity and functionality of retained organic structures. Collagen pull-off measurements not only support immunochemical evidence for collagen preservation for antibody recognition but also imply preservation of the whole molecular integrity. No appreciable differences in collagen pull-off properties were measured between fossil and extant bone samples under physiological conditions.

Introduction We have previously demonstrated by immunological methods that protein epitopes may survive across geological time in fossil materials that demonstrate morphological and histological integrity,1-3 and we have shown that antibody-antigen interactions can be used to identify and characterize organic components of fossils. However, measurable antibody-antigen interactions require only a few intact amino acids4 to show reactivity; hence, immunological methods alone do not provide information regarding the integrity or mechanical properties of protein fragments in fossil material. Atomic force microscopy (AFM) has the potential to complement and extend other analytical methods applied to fossils by determining the integrity and mechanical properties of organic molecules under physiological conditions, and indeed, AFM in combination with laser Raman spectroscopy has been applied to characterize Precambrian microfossils and shed light on the geochemical maturation of ancient organic matter.5 Here, we show that data obtained by AFM, either alone or in addition to other analytical tools, can be directly * Corresponding author. E-mail: [email protected]. Phone: (406) 994-6164. Fax: (406) 994-6040. † Montana State University. ‡ North Carolina State University. § North Carolina Museum of Natural Sciences. | Universitad Nacional del Comahue. (1) Schweitzer, M. H.; Watt, J. A.; Avci, R.; Knapp, L.; Chiappe, L.; Norell, M.; Marshall, M. J. Exp. Zool. 1999, 285, 146-157. (2) Schweitzer, M. H.; Watt, J. A.; Avci, R.; Forster, C.; Krause, D. W.; Knapp, L.; Rogers, R.; Beech, I.; Marshall, M. J. Vert. Paleo. 1999, 19, 712-722. (3) Schweitzer, M. H.; Marshall, M.; Carron, K.; Bohle, D. S.; Busse, S.; Arnold, E.; Johnson, C.; Starkey, J. R. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 6291-6296. (4) Child, A.; Pollard, M. J. Arch. Sci. 1992, 19, 39-47. (5) Kempe, A.; Schopf, J. W.; Altermann, W.; Kudryavtsev, A. B.; Heckl, W. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9117-9120.

compared with similarly treated material from related extant organisms and can be used to address the preservation of molecular function. Recently, exceptionally well-preserved avian eggs containing embryonic skeletal elements were described6 from exposures of the Bajo de la Carpa Member of the Upper Cretaceous Rio Colorado Formation in the city of Neuque´n, Argentina. The strata containing the eggs have been interpreted as interdune deposits corresponding to an eolian environment with fluvial influences, in a semiarid climate.7 Cladistic analysis of both the bones and the eggshell resulted in their assignment to a basal bird, most likely an enantiornithine.6 Thin and fragile embryonic bone tissues surrounded a calcite core that had precipitated within the original medullary space of the hollow long bones.6 The distinctive taphonomy and preliminary evidence of unusual preservation in gross and histological preparations of these embryonic tissues warranted further chemical and molecular analyses. Because collagen proteins are key components of virtually all vertebrate tissues and are extremely well characterized across a wide range of both tissues and taxa, we have selected this protein as a model system with which to demonstrate its morphological and unfolding properties. We have used a combination of high-resolution imaging and force-extension measurements and have compared the results with those of partially demineralized extant and fossilized bone tissues and nonmineralized (tendon) tissues. (6) Schweitzer, M. H.; Jackson, F. D.; Chiappe, L. M.; Schmitt, J. G.; Calvo, J. O.; Rubilar, D. E. J. Vert. Paleo. 2002, 22, 191-195. (7) Heredia, S.; Calvo, J. O. In Actas del XV Congreso Geologica Argentino; Cabaleri, N., Cingolani, C. A., Linares, E., Luchi, M. G. L. d., Panarello, H. O. O. y., Eds.; Calafate: Santa Cruz, Argentina, 2002; article 196197 (CD-ROM).

10.1021/la047682e CCC: $30.25 © 2005 American Chemical Society Published on Web 03/05/2005

Preservation of Late Cretaceous Bone Collagen

The vast majority of fossilized remains are those “hard” tissues resulting from biomineralization of organic matrixes during life, typically including bones and teeth. Interplay between protein and mineral in biomineralized vertebrate tissues is complex but invariably contributes to the potential of preservation in the fossil record. A better understanding of these natural “composite materials” is crucial to our understanding of a range of processes, from diagenetic changes occurring during molecular degradation to preservation of vertebrate remains over geological time spans. The diagenetic alteration of fossil tissues at the molecular level is not well understood; however, it has been shown that, in biomineralized tissues, proteinaceous matrix components may be stabilized and protected by association with (extrafibrillar) and incorporation into (intrafibrillar) mineral crystals.8-11 Atomic force microscopy (AFM) studies demonstrate that, in extant (bovine) bone, the ratio of extrafibrillar to intrafibrillar crystals is about 70-80% to 20-25%.12 In biomineralized tissues such as bones and teeth, charged amino acid residues within the protein component interact with hydroxylapatite mineral crystals to form a highly oriented, flexible, and strong composite. The organic matrix consists primarily of collagen (∼90%),13 a fibrillar protein composed of amino acids dominated by glycine, 4(R)-hydroxyproline, and proline residues. An individual type I collagen molecule, herein referred to as a collagen fibril, has a length of ∼300 nm and width of ∼1.4 nm14 and behaves like a flexible nanostring.15 The tertiary structure of the protein incorporated into mineralized tissues consists of three polypeptide chains in a helical arrangement, and recent AFM studies have provided further structural information by showing that, rather than being homogeneous, collagen fibers (a bundle of individual fibrils) are mechanically nonuniform.16 The “stacking” of helices to form the three-dimensional functional protein fibers results in a characteristic pattern of ∼67-nm bands, consistent across taxa and verified in ultramicrotomed sections of both tendon and bone collagen by AFM studies.12,13,17-19 The structure of collagen imparts unique properties to the molecule. One of these properties is for the molecule to act as a nucleus for mineral precipitation in biomineralized tissues. The ability of bone to resist sudden mechanical impact is attributed to so-called sacrificial bonds between collagen fibrils across the network of the composite material.18 These (sacrificial) bonds are attributed to inter- and/or intramolecular noncovalent bonds (8) DeNiro, M. J.; Weiner, S. Geochim. Cosmochim. Acta 1988, 52, 2415-2423. (9) Weiner, S.; Traub, W.; Elster, H.; DeNiro, M. J. Appl. Geochem. 1989, 4, 231-232. (10) Glimcher, M. J.; Cohen-Solal, L.; Kossiva, D.; Ricqles, A. d. Paleobiology 1990, 16, 219-232. (11) Sykes, G. A.; Collins, M. J.; Walton, D. I. Org. Geochem. 1995, 23, 1059-1065. (12) Sasaki, S.; Tagami, A.; Goto, T.; Taniguchi, M.; Nakata, M.; Hikichi, K. J. Mater. Sci.: Mater. Med. 2002, 13, 333-337. (13) Mbuyi-Muamba, J.-M.; Dequeker, J.; Gevers, G. Baillere’s Clin. Rheumatol. 1988, 2, 63-100. (14) Voet, D.; Voet, J. G. Biochemistry, 2nd ed.; John Wiley and Sons: New York, 1995; p 156. (15) Sun, Y. L.; Luo, Z. P.; Fertala, A.; An, K. N. Biochem. Biophys. Res. Commun. 2002, 295, 382-386. (16) Gutsmann, T.; Fantner, G. E.; Venturoni, M.; Ekani-Nkodo, A.; Thompson, J. B.; Kindt, J. H.; Morse, D. E.; Fygenson, D. K.; Hansma, P. K. Biophys. J. 2003, 84, 2593-2598. (17) Ottani, V.; Raspanti, M.; Ruggeri, A. Micron 2001, 32, 251-260. (18) Thompson, J. B.; Kindt, J. H.; Drake, B.; Hansma, H. G.; Morse, D. E.; Hansma, P. K. Nature 2001, 414, 773-775. (19) Avci, R.; Schweitzer, M.; Boyd, R.; Wittmeyer, J.; Steele, A.; Toporski, J.; Beech, I.; Arce, F. T.; Spangler, B.; Cole, K.; McKay, D. S. Langmuir 2004, 20, 11053-11063.

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forming cross-links that stabilize and protect the polymer backbone. No molecular-level understanding of the origin of the sacrificial bonds has been offered at this time; however, experimental evidence for their existence is irrefutable. The resilience and flexibility of bone is derived in part from these bonds breaking when stressed, thus dissipating stress energy in a nondestructive manner. Sacrificial bond breakage is measured by AFM and reflected in a characteristic saw-toothed pattern18 seen in AFM force-extension curves. These intermolecular bonds and interactions give collagen fibrils great tensile strength and increase its resistance to heat and chemical degradation,18 characteristics that make collagen a frequent target for molecular investigations of fossil specimens. Preliminary experiments suggested that collagen may be present in some elements of these Cretaceous embryonic bones. A comparison between these fossil and extant tissues using immunological and AFM techniques has been undertaken in the present study. Bone tissues from an extant neornithine hatchling bird (courtesy of J. Horner, Museum of Rockies, Bozeman, MT) were used for comparative analyses, and nonmineralized, paraformaldehyde-fixed tendons from extant chicken were similarly prepared and analyzed. Materials and Methods Tissue Preparation. Isolated chicken tendons were fixed in 4% paraformaldehyde in sodium-cacodylate buffer, rinsed with cacodylate buffer, and manually separated into individual fiber bundles. Small fragments of extant avian hatchling bone and fossil embryo bone were partially decalcified for 2-8 h in 0.5 M EDTA. All specimens were rinsed in sterile water, dehydrated in an ethanol series (30, 50, 75, 95, and 100%), and then equilibrated with LR White Hard Grade embedding resin (London Resin Company Ltd, Berkshire, U.K.) at 4 °C with 10 changes of resin. To achieve total infiltration, samples were placed under a vacuum for 24 h and then polymerized at 55 °C for 24 h. Sections (∼250 nm) were taken on a clean diamond knife using a Sorvall ultramicrotome and then heat-fixed at 55 °C to gelatin-coated glass microscope slides for immunohistochemical analysis or to 8-mm gelatin-coated circular glass or IR transparent Ge disks for AFM, Fourier transform infrared (FTIR), and time-of-flight secondary ion mass spectroscopy (ToFSIMS) analysis. A schematic is given in Figure 1. Immunohistochemistry and Collagenase Experiments. Bone sections (250 nm) were incubated with 0.5 M EDTA for 15 min to further decalcify, rinsed with a phosphate-buffered saline (PBS, 12 mM PO4- and 150 mM Na+), and etched over three 10-min incubations with 1 mg/mL sodium borohydrate. Sections were rinsed multiple times with 25 mM Tris, 5 mM CaCl2 (enzyme reaction buffer) and allowed to equilibrate in this buffer for 3 h. Following incubation, all sections were rinsed three times in enzyme reaction buffer, followed by being rinsed three rinses in PBS. The sections for immunohistochemical analysis were incubated in 4% normal goat serum (NGS) in PBS for 5 h to reduce nonspecific staining and were then incubated as follows: (1) test condition of purified polyclonal antiserum (10 µg/mL in 4% NGS) raised against chicken collagen I (Chemicon, AB752P); (2) 4% NGS not containing primary antibodies; (3) inhibited antibodies (500 µL of 10 µg/mL primary antibodies inhibited with 6.4 mg of lyophilized chicken collagen, 2.5 h at 21 °C); (4) sections were incubated for 3 h in collagenase enzyme (Sigma, C-1639) (1 mg/mL in enzyme reaction buffer), and controls were incubated in reaction buffer only. Sections were incubated with primary antibodies or controls for a duration of 12 h at 4 °C. Sections were then incubated sequentially with biotinylated Goat x-Rabbit IgG (1:500, Vector) for 1.5 h at 21 °C and Avidin-FITC (1:500, Vector) for 1.5 h at 4 °C (in the dark). Each incubation was followed by three rinses in PBS/0.1% Tween 20 and twice with PBS to remove unbound antibodies. For humic immunological experiments, a 5 mg/mL stock of humic acid in PBS (Fluka Chemika, 53680) was diluted to 0.5 and 0.25 mg/mL in pure water. Three 0.5-µL drops per well were

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Figure 1. AFM images obtained in air on 250-nm-thick sections of resin-embedded tissues. The schematic representation (center part) shows a sample embedded in resin and sectioned using an ultramicrotome to 250 nm. The AFM images obtained in air are the following: (A) Embedding resin with no specimen. The sectioning marks are clearly visible, but resin is otherwise featureless. (B) Partially decalcified extant bone. The fibrillar nature of collagen matrix is apparent but somewhat obscured by overlying mineral. The 67-nm banding pattern characteristic of collagen I is visible (e.g., see arrow) on individual fibers. (C) Fossil bone, visualized under identical parameters. Scattered fiberlike features are visible among the wavy texture, but banding is not discernible. (D) Extant chicken tendon, as above. The 67-nm banding pattern is obvious across the tendon. heat-fixed to PTFE-coated microscope slides (EMS, 63419-08) and then treated with antisera or controls as described above. Cover slips were applied using Vectashield mounting media (Vector), and sections were imaged using a compound microscope (Zeiss, Axio Skop2) equipped with a FITC filter. Exposure times ranged from 150 to 700 ms. For AFM collagenase experiments, ∼150 µL of collagenase (with a concentration of ∼2.5 mg/mL in enzyme reaction buffer) was pipetted onto the embedded sections and allowed to incubate

Avci et al. for ∼24 h prior to washing, drying, and imaging. Following incubation, the sections were rinsed five times in PBS and imaged in air using tapping mode AFM. Humic Sample Preparation. In addition to immunological controls, FTIR and AFM pull-off experiments were conducted on complex humic substances (Fluka Chemika, 53680). For FTIR experiments, a solution of humic substances was prepared at an ∼1 mg/mL concentration in pure water, and ∼400-µL drops were spotted onto IR transparent Ge disks (WJD-U25, Harrick Sci. Co., Broadway, NY) and dried overnight. Additionally, 250-nm sections of fossil and extant bone, chicken tendon, and embedding resin were placed directly on the Ge disks and allowed to dry thoroughly before being subjected to FTIR experiments. Absorbance was determined using an FTIR spectrometer (Nicholet, model 740) in transmission mode. Humic complexes were immobilized on amino-functionalized Si3N4 wafers by the following procedure: ∼400 µL humic solution was diluted in 0.1 M MES (containing 0.5 M NaCl, pH 6.0) and mixed with 0.4 mg of EDC and 0.6 mg of NHS. This mixture was allowed to react for 15 min at room temperature before 1.4 mL of 2-mercaptoethanol was added. The amino-functionalized wafers were incubated in this solution for 2 h at room temperature and then transferred to a beaker containing 1 mL Tris (20 mM, pH 7.3). The wafers were rinsed four times with deionized water and air-dried as described above for bone and tendon sections. AFM Measurements. All AFM images were taken using a Multimode nanoscope IIIa system, equipped with a vertically engaged J-scanner. Si cantilevers (TAP3000 HD, NanoDevices, Santa Barbara, CA) having nominal resonance frequencies of ∼300 kHz and spring constants of ∼40 N/m were used to obtain air tapping mode images (Figure 1) from a 4 × 4 µm2 area of tissue embedded and sectioned as described above. After obtaining images in air, the tip was replaced with a silicon-nitride tip (Veeco Metrology) with a spring constant of 0.01 N/m, and a contact mode image of the bone section was performed in air with the soft tip to ensure that the 4 × 4 µm2 area previously identified by the stiff tip was not lost. In some cases, a Dimension 300 AFM system (Veeco Metrology) was used to obtain AFM tapping mode images in air to accommodate samples larger than the Multimode instrument is able to handle (∼1 cm diameter). Finally, PBS (100 µL) was injected into the liquid cell and incubated for 1 h to hydrate and equilibrate tissues before AFM measurements in liquid. Force-volume measurements were conducted over the 4 × 4 µm2 area divided into 32 × 32 pixels. Each force curve was obtained with a 1-Hz frequency and had a z-scan range of 500 nm and a maximum deflection set point of 50 nm (corresponding to a maximum load of 0.5 nN). After force measurements, AFM images in liquid (either contact or tapping mode) were obtained to ensure that the bone section under study was intact. FTIR and ToFSIMS Analysis. For FTIR analysis, fossil bone sections and similarly prepared sections of chicken tendon, extant bone, and resin were placed on IR transparent Ge disks and examined in the spectral range from 800 to 1800 cm-1 (amide band region). To give support to our conclusion that fossil material contains organic substances, these sections were analyzed using imaging time-of-flight secondary ion mass spectroscopy (ToFSIMS) in order to assess the organic and inorganic material content and distribution of these sections. No special sample preparation was needed for these studies: the same sample that was used in the FTIR studies worked excellently for ToFSIMS analysis. We employed the PHI-EVANS’s TRIF I ToFSIMS system housed at the Image and Chemical Analyis Laboratory at Montana State University (www.physics.montana.edu/ical). Statistical Analysis. A MatLab program was used to analyze the force curves obtained by the force-volume technique. Each pull-off curve was analyzed by first finding the zero force line; the intersection of this line with the pull-off curve (blue curves in Figure 3) identifies the tip contact point in the blue curve. This point corresponds, for example, to ∼72 nm in extant bone (Figure 3B). The MatLab program identifies the unbinding events by searching the local minima.20 For example, the second event (20) Arce, F. T.; Avci, R.; Beech, I.; Cooksey, K.; WigglesworthCooksey, B. Biophys. J. 2004, 87, 4284-4297.

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Figure 3. Force curves obtained on sections of (A) fossil embryo bone, (B) extant bird bone, (C) resin, and (D) tendon. The green lines correspond to tip approach and the blue lines to pull-off curves. Sharp minima (e.g., arrow in part B) mark the unbinding events. The more gradual slope of the approach curve seen in fossil bone after tip-surface contact suggests that fossil bone is more flexible (“softer”) than other surfaces examined, consistent with partial degradation of collagen bone matrix. In some cases, the fossil bone surface shows elastic degradation, indicated by a separation in the blue and green lines during tip contact. The red dotted lines correspond to slopes of the force curves just before unbinding.

Results and Discussion

Figure 2. Immunohistochemical images obtained using optical fluorescence microscopy on partially demineralized 250-nm sections of extant hatchling (A, C, E, and G) and fossil embryonic (B, D, F, and H) avian bone: (A and B) Sections are incubated with primary antibodies against avian collagen I followed by secondary antibodies conjugated with a fluorescent label. Osteocyte lacunae are marked by white ellipses. The scale bars in each part represent 20 µm. The green color marks the locations of reactive collagen epitopes, demonstrating that the immunological response of fossil collagen is similar to that of extant collagen. (C and D) To demonstrate the specificity of primary antibody binding, anti-collagen antibodies were blocked by incubating first with collagen and then exposed to bone sections. (E and F) To demonstrate that collagen proteins are the target of the antibodies, sections were digested with the collagen-specific enzyme collagenase and then incubated with primary and secondary antibodies as in parts A and B. (G and H) To control for nonspecific binding of secondary antibodies, sections were incubated with secondary antibodies only, and no primary anti-collagen antibodies were added. All other parameters were identical to parts A and B, and the imaging conditions were identical in all cases. from the left in the extant curve (marked by an arrow in Figure 3B) appears at ∼208 nm at ∼ -0.52 nN. The -0.52 nN corresponds to ∼52 nm of cantilever deflection. From these data, the collagen extension is then calculated by subtracting the cantilever deflection and the displacement associated with the contact point from the displacement associated with the particular unbinding event; the example here yields L ) 208 - 72 - 52 ) 84 nm. The unbinding force, calculated as the difference between the forces corresponding to the minimum (-0.52 nN) and the maximum to its right (-0.34 nN), yields an unbinding force value of -0.18 nN. Only those forces greater than 0.025 nN were considered to distinguish unbinding events from instrument noise.

To maximize the number of results and controls working with a very small amount of fossil material, we conducted experiments on sections that varied in thickness from 200 to 300 nm, with tissue sizes ranging from 100 to 200 µm in diameter. The immunohistochemical work with optical fluorescence microscopy and AFM studies uses similarly prepared sections. Here, we introduce the schematic of a typical thin section, depicted in the center part of Figure 1. The other parts, labeled as A, B, C, and D in Figure 1, correspond to the morphological images associated with resin, extant bone, fossil bone, and tendon sections, respectively. The discussion of these samples and their morphologies is deferred to the AFM Results section below. Immunohistochemical Results. In situ immunohistochemical analyses identified antigenic material within samples. Scanning confocal laser microsocopy (SCLM, not shown) and optical fluorescence microscopy (Figure 2) were used to monitor antibody binding on the labeled sections. Antibody binding was observed in both extant and fossil bone exposed to collagen antiserum (green patches in Figure 2A,B). The fluorescent signal corresponding to antibody-antigen interactions was significantly greater than that seen in the negative controls. When collagenspecific antibodies were first incubated with excess avian collagen to block binding sites on collagen antibodies and then incubated with sections as described, the fluorescent signal was completely blocked, testifying to the specificity of antibody-antigen interactions in these samples (Figure 2C,D). When sections were subjected to digestion with collagenase and then incubated as described for Figure 2A,B, the immunological signal was significantly reduced (Figure 2E,F). Finally, sections incubated with no primary antibody, but otherwise identical in treatment with the test conditions, show that the fluorescent signal is not due to nonspecific interaction of secondary antibodies with

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tissue but dependent upon interactions of the anti-collagen antibodies with epitopes preserved within the tissue sections (Figure 2G,H). Osteocyte lacunae, delineated by white circles on the images of both the extant and fossil bone sections (Figure 2A,B), indicate that antibody binding in both cases is localized to the bone matrix and is not the result of antibody interactions with embedding resin. However, in some regions of the fossil bone, the fluorescent signal was more uneven than that in extant samples, indicating a differential and patchy preservation of fossil bone epitopes consistent with partial degradation/ alteration of organics in some regions of the tissue. AFM Results. A schematic illustration of section preparation and representative images is shown in Figure 1. Images obtained using AFM in air clearly show a fibrillar pattern with characteristic cross-banding in extant samples (Figure 1B,D), although in extant bone the pattern is less distinct than that in tendon fibers, due to uneven masking of overlying biominerals remaining after partial demineralization. Images from fossil embryo bone show fiberlike features distributed more or less homogeneously over a wavy texture (Figure 1C), but cross-banding is difficult to assess. These features are possibly due to partial degradation/alteration of fossil collagen fibers. Resin morphology (Figure 1A) showed no distinctive pattern except sectioning artifact; therefore, tissues and embedding resin could be easily differentiated by both optical and AFM imaging in liquid, verifying that force-extension data were obtained from the regions of interest. Furthermore, ToFSIMS analysis of these sections (not shown) demonstrates clearly that resin material did not diffuse into the fossil matrix and that the organic signature of the fossil, in terms of fragmentation patterns, is very different from that of the resin. After images were obtained in air, samples were immersed into physiological buffer (PBS) in a liquid AFM cell and force-extension measurements were obtained on fossil and extant bone sections to determine the pulloff properties of the protein matrix. Figure 3 represents force-extension curves obtained on sections of fossil bone (Figure 3A), extant bone (Figure 3B), resin (Figure 3C), and extant tendon (Figure 3D). The saw-toothed features, herein called unbinding events or just events (an example is marked by an arrow in Figure 3B), seen in the forceextension curves are due to one of the three following possibilities: (1) the detachment of the tip from the surface (a nonspecific short-range event), (2) the detachment of a single fibril (individual collagen molecule) stretched between the surface and the tip, or (3) the rupture of interor intramolecular bonds (relaxation of sacrificial bonds) associated with a single fibril, causing a sudden reduction of load on the atomic force microscope tip. The concept of sacrificial bond rupture was first introduced and used by Hansma et al.,18 and here, we adopt their interpretation. To assess the similarity of force curves, slopes were calculated for the curves near the tips of the saw-toothed patterns and are represented by red dotted lines in Figure 3. The absolute value of the slope obtained on the fossil embryonic bone (4.0 ( 0.7 pN/nm) was similar to those obtained on extant bone (5.1 ( 0.8 pN/nm) and tendon (4.0 ( 0.8 pN/nm) and clearly distinct from values obtained on the resin control (7 ( 1.5 pN/nm), indicating similarities in mechanical properties among the biological samples. Unembedded fossil bone immobilized on a mica surface using a commercial epoxy (Devcon, Rivieray Beach, FL) yielded curves very similar to those in Figure 3A, supporting the evidence that the observed unbinding events are intrinsic to fossil bone. All force-extension curves show multiple unbinding events (arrow, Figure

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3A), corresponding to sudden changes in the tip equilibrium as it returns closer to the zero force line. In the examples shown in Figure 3, the extant bone sample (Figure 3B) shows six separate events in the forceextension curve, while five events are obtained from fossil bone (Figure 3A), suggesting similarities in inherent molecular pull-off properties. The contact regions of the force curves (upward bend in the green/blue curves in Figure 3) are sensitive to the microelastic properties of the surface.21 For example, the pull-off (blue) curve derived from fossil bone lags behind relative to the approach (green) curve in the contact region (Figure 1A), suggesting plastic deformation and hence moderate degradation of elastic properties in the Cretaceous sample. However, in some cases, approach and pull-off curves overlapped perfectly with no degradation in elastic properties. Taken together (Figures 1-3), these data support the hypothesis that the material in the fossil bone is organic and consistent with collagen in molecular unfolding properties and antigenic response. The conclusion that the fossil section is organic is further supported by ToFSIMS analysis (not shown) and the collagenase digestion experiments presented below (Figure 7). Imaging ToFSIMS shows an abundance of organic fragments generated from the fossil section mixed with inorganic components dominated by calcium. A full comparison of the statistical distributions of unbinding events in fossil and extant sections requires further analysis. Using a MatLab-based analysis,20 1200 unbinding events were identified in the extant bone and 1711 events were identified in the fossil. It is important to note that the number of events varies from sample to sample. In some cases, we observed less than 1000 events. The statistics discussed here pertain to the particular force-volume data presented here. In this case, 18 out of 1024 force curves obtained from the extant bone and 5 from the fossil were identified as corrupt curves and are therefore not included in the analyses. The force curves obtained for a given section are uniformly distributed over a 4 × 4 µm2 area (force-volume data). The events in each pull-off curve have been classified as no event, single event, or multiple events based on the profile obtained for each. The distribution of these events depends partly on the quality of the section under study and partly on the sample itself. For example, for the sample studied here, approximately 30% of the curves obtained on extant bone showed no events, 38% showed single events, and 32% showed multiple events. On the other hand, for the distribution of curves obtained on the fossil bone, 13% showed no events, 36% showed single events, and about 51% showed multiple events. Figure 4 compares the magnitude of the unbinding force and the corresponding extension for each event for extant (black points) and fossil (red points) bone. Data from extant sections show distributions of greater forces (some larger than 0.6 nN) concentrated around 100-nm extensions, while those from fossil bone show force distributions of as much as 0.4 nN with extensions exceeding 150 nm. The variations in magnitude of the unbinding forces suggest multiple collagen attachments to the atomic force microscope tip, where subsequent detachments of individual fibrils plus the unbinding of sacrificial bonds give rise to the observed force-extension profiles. We attribute most of the events with unbinding forces