Methods for Studying Fossil Molecular Histology
Examination of fossil/sub-fossil molecular histology is proposed for empirically studying how the cumulative effect of diagenetic variables upon a specimen’s molecular histology correlates with degree of sequence preservation. Vertebrate elements with the highest potential for molecular sequence preservation include tooth enamel and dentine, bone, and eggshell3, 5, 59. Of these, bone by far is the most widely characterized within ancient specimens as to its non-mineral histological structures. Numerous studies have reported histological structures morphologically and chemically consistent with biological cells, vascular tissue, and “collagenous” matrix preserved within Cenozoic and Mesozoic bones22, 28-40. In particular, the organic portion of extant collagenous bone matrix is comprised of ~90% type-1 bone collagen40, 59. This high proportion of a single, specific molecule is practical for comparison against purified collagen standards, extant controls, and across various ancient specimens.
The above histological structures are generally isolated via demineralization using a dilute acid31, 33, 36; this allows their molecular histology to be investigated using a suite of molecular methods. Characterization of morphology for these structures has historically been accomplished using a combination of light microscopy and both of transmission and scanning electron microscopy31-33, 35, 36, 41. Light microscopy is a practical method to rapidly screen specimens for the preservation of histological structures. The use of both transmission and scanning electron microscopy together is particularly advantageous. While both offer nano-scale optical resolution, the former images sample cross-sections while the latter sample surface63, 64. Both methods are also readily capable of detecting a distinct ~67nm banding pattern unique to collagen protein helices40, 65-67. Observation of this banding pattern indicates either the presence of a collagen helix or compounds replicating its structure.
Studying the chemical aspect of molecular histology generally requires localizing chemical signal to a specific histological structure. Two methods with precedence for use within molecular paleontology are time-of-flight secondary ionization mass spectrometry (ToF-SIMS) and Raman spectroscopy:
ToF-SIMS rasters a microscale-diameter ion beam in a square, grid-like pattern across a specimen’s surface. At each point in the square analysis “grid”, the chemical content of the specimen at that specific point is detected and recorded as a spectrum of molecular and fragment ions. A specific ion can then be plotted according to its recorded intensity at each point in the grid to form a molecular map that mirrors the area analyzed across the specimen’s surface. The specific types of ions detected via this process vary depending upon specimen chemical makeup; this allows the unique histological structures of a specimen to be targeted so that chemical makeup can be connected to morphology68-70. A few studies have employed ToF-SIMS to analyze ancient specimens29-31, 36, 52, 71-73. One recent publication used the method to analyze the molecular histology of demineralized epidermis from an exceptionally preserved Jurassic ichthyosaur31. Ionic fragments consistent with peptides or related compounds, along with polyaromatic hydrocarbons, were successfully localized to the ichthyosaur epidermis. Recorded intensities for polyaromatic hydrocarbon and peptide-related ion fragments (such as those detected in the Jurassic ichthyosaur31) can be compared across extant and ancient histological structures. For example, elevated levels of polyaromatic related ions in one specimen relative to another would be predicted to indicate a higher degree of chemical degradation68, 74-77. This is one potential method for evaluating changes in fossil/sub-fossil molecular histology by geologic timepoint and depositional environment.
Raman spectroscopy utilizes a monochromatic laser to irradiate a single point a few microns in diameter on a specimen surface78-80. As the laser’s photons contact the specimen surface, a small number of them are inelastically scattered by the specimen surface; that is, they either gain or lose energy after contacting the specimen surface81, 82. The degree to which these photons change energy depends on the type of molecular bond vibration the photon contacted within the specimen. Detecting the change in these photons’ energies forms a spectrum revealing the types of molecular bond vibrations present where the laser contacted. This allows specific histological structures to be analyzed for the types of molecular bonds present in their chemical makeup78-80, 83. A recent study attempted to analyze the molecular histology of fossil tissues using Raman spectroscopy22. However, perusal of their published findings raised questions as to whether some of their data represented true Raman signal or was an artifact of autofluorescence84. Raman spectroscopy with a laser wavelength below 250nm is a well-established solution to eliminate autofluorescence42, 85 but has seen little use within molecular paleontology historically42. However, similar to the ion intensities with ToF-SIMS, Raman signal intensity for specific bond vibrations can be compared across extant and ancient specimen molecular histology. Indeed, this method has seen substantial use historically in correlating thermal history with molecular makeup for a wide range of humics and kerogen macromolecules in petroleum and soil science76, 86, 87.
Data collected using these described techniques can be correlated with the degree to which molecular sequences are recoverable from fossil and sub-fossil specimens. Both the intensity of Raman signal for specific bond vibrations and the relative ion abundances from ToF-SIMS can readily be compared against the degree to which a specimen preserves molecular sequence information. In the case of collagen peptides, both forms of electron microscopy can be used to evaluate the relative abundance of ~67nm banding present within bone matrix. This too can be compared against the degree of type-1 collagen sequence information recoverable from a given specimen.