Casey Theriot
Feb 3, 2010
Micro Journal Club
Introduction
Jurassic Park
Molecular Paleontology
Molecular: relating to or produced
by or consisting of molecules
Paleontology: the study of prehistoric
life, including organisms’ evolution and
interactions with each other and their
environments.
John Horner, MSU
Mary Schweitzer, NCSU
Bone lingo
Trabecular: Spongy bone, found at the
end of long bones. Porosity is 50-90%
Cortical: Compact bone found in shaft
of long bones, forms around trabecular.
Denser porosity, 5-10%
Osteocytes: star shaped cell, most
abundant in compact bone networked by
long cytoplasmic extensions called
lacunae. They are involved in turnover
of bone matrix, or destroying bone.
Hydroxyapetite
(Ca
carbonate/Ca
phosphate) is deposited around bone
too)
1. Clusters of spheres showed an iron-oxygen
elemental signal and appeared red under light
microscope
2. Soft tube-like structures that contain spheres
3. Free-floating osteocytes with filipodia
4. A filamentous mass that remained pliable and
elastic
ostrich
ostrich
Fig. 1. Demineralized fragments of endosteally derived tissues lining
the marrow cavity of the T. rex femur. The demineralized fragment is
flexible and resilient and, when stretched (arrow), returns to its original
shape. (B) Demineralized bone in (A) after air drying. The overall
structural and functional characteristics remain after dehydration.
(C) Regions of demineralized bone show fibrous character (arrows).
Scale bars, 0.5 mm.
Fig. 2.
A.
B.
C.
D.
E.
F.
G.
H.
Demineralization of cortical bone reveals the presence of soft tissue structures
Partial demineralization of T-rex cortical bone. Shows network of vascular canals
Second sample shows transparent vessels arising from bone matrix
Complete demineralization, transparent flexible vessels in remains of cortical bone
Ostrich vessel after demineralization of cortical bone and digestion of collagenous matrix
Higher magnification of T-rex vessels, red spheres are present, 50 um
T-rex vessel with red sphere, compare to ostrich vessel in H
Second fragment of T-rex
Ostrich vessel digested from demineralized cortical bone. Red blood cells are seen here
Fig. 4. Cellular features associated with T. rex and ostrich tissues. (A) Fragment of
demineralized cortical bone from T. rex, showing parallel-oriented fibers and cell-like
microstructures among the fibers. The inset is a higher magnification of one of the
microstructures seen embedded in the fibrous material. (B) Demineralized and stained
(3) ostrich cortical bone, showing fibrillar, parallel-oriented collagen matrix with
osteocytes embedded among the fibers. The inset shows a higher magnification of one
of the osteocytes. Both inset views show elongate bodies with multiple projections
arising from the external surface consistent with filipodia. (C) Isolated microstructure
from T. rex after fixation. In addition to the multiple filipodial-like projections, internal
contents can be seen. The inset shows a second structure with long filipodia and an
internal transparent nucleus-like structure. (D) Fixed ostrich osteocyte; inset, ostrich
osteocyte fixed and stained for better visualization. Internal contents are discernible,
and filipodia can be seen extending in multiple planes from the cell surface. (E and F)
SEM images of aldehyde-fixed (3) microstructures isolated from T. rex cortical bone
tissues. Scale bars in (A) and (B), 50 µm; in (C) and (D), 20 µm; in (E), 10 µm; in (F),
1 µm.
T. rex
Ostrich
T. rex
Ostrich
T. rex
T. rex
Hypothesis
•  An exceptionally well-preserved bone
may act as a containment vessel for
biomolecules
Table 1: Specimens examined
2.588m – 12000
55.8 – 33.9 Ma
65.5 – 55.8 Ma
145.5 – 65.5 Ma
Most
recent
Old
Really old
Really
really
old
This study covers seven geologic
formations and 15 genera.
Formation: fundamental unit of
Lithostratigraphy.
Lithostratigraphy:
sub-discipline
of stratigraphy, geological
science associated with the
study of strata or rock layers.
Part 1: Figure 1
Framboidal structure
comprises roughly
spherical aggregates
of discrete equiregular euhedral
microcrystallites of
around 0.5µm in
diameter, with the
average aggregate size
ranging from 5-20µm.
Energy dispersive X-ray spectroscopy (EDS) is an analytical technique used for the elemental
analysis or chemical characterization of a sample.
Figure 2
Well preserved turtle phalange used.
Figure 3
www.spie.org
SEM of dinosaur trabecular bone
Calling this iron oxide framboid cluster
10 um diameter
catalog.nucleusinc.com
Ammonite suture has framboids too
Part 2: Figure 4
Framboids?
Figure 5
Arrows identify coatings that peeled
away from the bone when fractured,
revealing a layered structure.
Figure 6
Typical vascular canal
Elemental map from EDS
Iron
Calcium
Iron mineralization in vascular canal
Figure 7
Framboid surface
Vas. Canal surface
Framboids with bubble like
structure or biofilms covering
them
Bubble like structures
Vascular canal of trab. bone
Figure 8
cracks
The cracks are formed by free swimming microbes
or bacteria in a viscous medium-again reinforcing
the biofilm hypothesis.
trough
Higher magnification which shows troughs
Bridging trough structures
Figure 9
Infrared spectral comparison: 83% match between fossil and biofilm, with
37% correlation to collagen
Part 3: Figure 10
Osteocytes found after acid wash
Pre-acid wash of lacunae
SEM pics of lacunae
Part 4: 14C Dating
•  Results: Material removed from the
vascular canal “Greater than modern”
•  Greater than 1950
•  Usually BP or before present
Discussion
•  Iron-Oxygen spheres are framboids and are too
common to be extraordinary preservation
•  Bubbles suggest release of gases- suggests biofilms
•  Tubular structure and osteocytes can mimic biofilm
structure or endocasts
•  Structures are a product of common bacterial
activities
•  Where are the SEM images of the control Biofilms
that they grew?
Do you believe this?
Theories?
•  Biofilms
•  Biomineralization is occuring
•  Bacteria are playing a role by helping
preserve the soft tissue/biomolecules
•  Nanobacteria are present
•  Artifacts from SEM
SEM biofilm preps
ESEM sample;
water vapor pressure = 5.5 torr
SEM sample preserved by air-drying
SEM sample prepared by ethanol
dehydration with HMDS drying.
SEM sample dried under vacuum
SEM sample prepared by 10%
glutaraldehyde fixation and air drying.
SEM sample prepared by ethanol
dehydration with critical-point drying.
Fratesi, SE et al. Effects of SEM preparation techniques on the appearance
of bacteria and biofilms in the Carter Sandstone. 2004.