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.