Three-dimensional visualization of fossil flowers, fruits,
seeds, and other plant remains using synchrotron
radiation X-ray tomographic microscopy (SRXTM): new
insights into Cretaceous plant diversity
Authors: Friis, Else Marie, Marone, Federica, Pedersen, Kaj
Raunsgaard, Crane, Peter R., and Stampanoni, Marco
Source: Journal of Paleontology, 88(4) : 684-701
Published By: The Paleontological Society
URL: https://doi.org/10.1666/13-099
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THREE-DIMENSIONAL VISUALIZATION OF FOSSI L FLOWERS, FRUITS,
SEEDS, AND OTHER PLANT REMAINS USING SYNCHROTRON RADIATION
X-RAY TOMOGRAPHIC MICROSCOPY (SRXTM): NEW INSIGHTS INTO
CRETACEOUS PLANT DIVERSITY
ELSE MARIE FRIIS,
1
FEDERICA MARONE,
2
KAJ RAUNSGAARD PEDERSEN,
3
PETER R. CRANE,
4
AND MARCO STAMPANONI
2,5
1
Department of Palaeobiology, Swedish Museum of Natural History, SE-104 05 Stockholm, Sweden, ,else.marie.friis@nrm.se.;
2
Swiss Light Source, Paul Scherrer Institute, CH-5232 Villigen PSI, Switzerland, ,federica.marone@psi.ch.; ,marco.stampanoni@psi.ch.;
3
Department of Geology, University of Aarhus, DK-8000 Aarhus, Denmark, ,krp@geo.au.dk. ;
4
Yale School of Forestry and Environmental Studies
195 Prospect Street, New Haven, CT 06511, USA, ,peter.crane@yale.edu.; and
5
Institute for Biomedical Engineering, ETZ F 85,
Swiss Federal Institute of Technology Zu¨rich, Gloriastrasse 35, 8092 Zu¨rich, ,stampanoni@biomed.ee.ethz.ch.
ABSTRACT—The application of synchrotron radiation X-ray tomographic microscopy (SRXTM) to the study of mesofossils
of Cretaceous age has created new possibilities for the three-dimensional visualization and analysis of the external and
internal structure of critical plant fossil material. SRXTM provides cellular and subcellular resolution of comparable or
higher quality to that obtained from permineralized material using thin sections or the peel technique. SRXTM also has the
advantage of being non-destructive and results in the rapid acquisition of large quantities of data in digital form. SRXTM
thus refocuses the effort of the investigator from physical preparation to the digital post-processing of X-ray tomographic
data, which allows great flexibility in the reconstruction, visualization, and analysis of the internal and external structure of
fossil material in multiple planes and in two or three dimensions. A review of recent applications in paleobotany
demonstrates that SRXTM will dramatically expand the level of information available for diverse fossil plants. Future
refinement of SRXTM approaches that further increases resolution and eases digital post-processing, will transform the
study of mesofossils and create new possibilities for advancing paleobotanical knowledge. We illustrate these points using
a variety of Cretaceous mesofossils, highlighting in particular those cases where SRXTM has been essential for resolving
critical structural details that have enhanced systematic understanding and improved phylogenetic interpretations.
INTRODUCTION
T
HE PAST thirty to forty years have seen significant a dvances
in understanding patterns of str uctural diversification
during the early phases of angiosperm evolution. Central i n
these developments has been the discovery in Cretaceous
sediments of small 3-D ve getative and reproductive struc tures
(mesofossils), including angiosperm flowers, fruits, and seeds
(Tiffney, 1977; Friis and Skarby, 1981; see also referenc es in
Friis et al., 2011) . The se f ossils, which are preserved both as
lignitized specimens and as charcoal, often have e xquisite
preservation of complex form as well as superb preservation of
cellular and other internal details. Initially, this material was
studied with considerable success using scanning electron
microscopy (SEM), occasionally supplemented by conventional
serial sectioning after embedding the specimens in p lastic. Ove r
time technical advances in SEM also improved resolution in the
routine microscopy of surface features.
More recently, the application of synchrotro n radiat io n X-ray
tomographic microscopy (SRXT M) has provided a new way of
studying diverse mesofossils through the digita l capture of high-
resolution, large, X-ray tomographic, datasets. Unlike with
conventional sectioning, these datasets are created withou t
damage to the specimen, and they enable the rec onstruction,
visualization, and analysis of the internal and external structure
of critical fossil material with new flexibility. Two-dimensional
sections can be constr ucted in multiple orientations, 3-D
reconstructions can be created and manipulated, and complex
specimens, such as flowers, can be dissected digitally rather
than physically. S RXTM gre atly e nhances the information that
can be o btained from fossil plants for comparative and
phylogenetic studies (Friis et al., 20 07).
Extensive datasets on fossil plants have been accumulated
from SRXTM analyses at the Tomcat beamline (Stampanoni et
al., 2006) a t the Swiss Light Source (SLS) covering material
from the Carboniferous (Scott et al., 2009), Permian (Slater et
al., 2011), Cretac eous (Friis et al., 2007; von Balthazar et al.,
2007; von Balthazar et al., 2008; Friis et al., 2009b; Friis et al.,
2010; Friis and Pedersen , 2011; He
ˇ
rmanova
´
et al., 2011; von
Balthazar et al., 2011; Friis and Pedersen, 2012; Friis et al.,
2013a; Friis e t al., 2013b; Friis et al., 2014a, 2014b; Mendes et
al., 2014), and Cenozoic (Smith et a l. , 2009a; Smith et al.,
2009b; Collinson et al., 2013 a; Collinson et al., 2013b). The
only other published studies on Cretace ous flowers examined at
other beamlines is that of Glandulocalyx upatoiensis Scho
¨
nen-
berger et al., 2012, analyzed mainly at th e b eamline 2-BM of the
Advanced Ph oton Source at the U.S. Department of Energy
Argonne National Laboratory, but also at the BL20B2 beamline
of the Super Proton ring-8 GeV (SPr ing-8) at the Japan
Synchrotron Radiation Research Institute (Scho
¨
nenberger et
al., 2012), and unnamed flower studied at the beamlines BM05,
ID19, and ID22 at ESRF, Grenoble (Mor eau et al., 2014). There
are also now several studies of extant floral structur es using
laboratory based X-ray CT (e.g., Staedler et al., 2013).
In this paper we prov ide an overview of the SRXTM
techniques applied so far to understand and visualize the
detailed structure of Cretaceous fossil flowers and other plant
mesofossils. We focus on examples from the very substantial
datasets collected a t the SLS and highlight those cases where
SRXTM has resolved critical structural details that have
684
Journal of Paleontology, 88(4), 2014, p. 684–701
Copyright Ó 2014, The Paleontological Society
0022-3360/14/0088-684$03.00
DOI: 10.1666/13-099
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improved systematic und erstanding and phylogenetic interpre-
tation.
FOSSIL MATERIAL
The first substantia l discovery of a Cre taceo us mesofossil
assemblage c ontaining well-preserved flowers w as from fluvi-
atile-lacustrine sediments of late Santonian to early Campanian
age at the
˚
Asen locality, Southern Sweden (Friis and Skarby,
1981). Since the n many similar floras have been disc overed and
studied from localities of Creta ceous age in Europe, North
America, Central and East Asia, Antarctic a, and New Zealand
(for referenc es se e Friis et al., 2011). The Cretaceous
mesofossils occur as iso lated organs in unconsolidated clays
and sands, and are extracted by sieving in water. Intermediate in
size between the larger fossils (macrofossils) that have typically
been the focus of Cr etaceous paleobotanical re search, and fossil
pollen and spores (microfossils), the flowers, fruits, seeds an d
other fossils that comprise mesofossil assembla ges rare ly exc eed
more than a few millimeters in length.
Fossil assemblages containing isolated plant fra gments are
common in Cenozoic strata (e.g, Reid and Reid, 1915; Chandler,
1957; Kirchheime r, 195 7; Dorof eev , 1963; Friis, 1985; see also
references in Mai, 1995), and have also bee n obtained from
older sediments (Edwards, 1996; Crane and Herendeen, 2009),
but sever al features of many Cretaceous mesofossil a ssemblages
are unusual. In particular, the small size of the individ ual fossils,
and the presence of large numbers of tiny fossil f lowers, often
with delicate petals, s tamens and other floral parts preserved,
was completely unanticipated. Also unusual is the abundance of
charcoal in these Cretaceous mesofossil assemblages. Compa-
rable assemb lages of Cenozoic age, prepared using the same
techniques, typically contain fossils with a much wider range of
sizes (Tiffney, 1984; Eriksson et al., 2000a), are usually
preserved as lignite rather than cha rcoal, and rarely contain
fossil flowers (Friis et al., 2011).
The abundance of charcoal in Cretac eous mesofossil assem-
blages indicates t hat na tural fires were a major fea ture of
Cretaceous landscapes (Friis et al., 2011; Brown et al., 2012). In
the process of c harcoalif ication , the incomplete combustion of
plant ma teria l under conditions of reduced oxygen resulted in
excellent preservation of the 3-D form and cellular detail of
diverse plant parts. While some shrinkage of the original plant
material often occurs during charc oalification (Harris, 1981;
Lupia, 1995), and cell walls are typically homogenize d, the
shape of the cells is gener ally more or le ss unaltere d (Scott and
Jones, 1991), and very delicate structural details are fre quently
preserved, as illustrated in the descriptive part of this revie w.
The 3-D preservation of exquisite cellular details, combined
with their small size, makes ma ny fossil flowe rs and other
reproductive structures from Cretaceous me sofossil floras
especially well-suite d for SRXTM. The application of the new
technique to charcoalified material has allowed mesofossils to
be examined with an unprecedented level of detail and is
advancing our unde rstanding of Cretaceous plant diversity in
substantial ways.
SRXTM TECHNIQUES
Synchrotron radiation har d X-ray tomographic microscopy
(SRXTM) represents a great advance over the application of
conventional X-ray approaches in paleontology and has proved
a powerf ul technique for the non -destructive investigation of
internal structure in a variety of optically opaque samples.
Broadly similar SRXTM tec hniques c an b e ap plied to a wide
range of paleobiological ma teria l, but here we focu s on the
techniques used so far f or the study of plant mesofossils.
Preparation of plant mesofossils for SRXTM is straightfor-
ward. Specimens are analyzed non-destructively and are n ot
altered physica lly by the process. Unlike SEM no coating is
required, and unlike with c onventi onal sec tioni ng of livi ng
material dehydrating, f ixing or staining of the specimen is not
needed. The specimen is usually attached to a support (e.g., a
brass pin or SEM stub) with a diameter compatible with that of
the beamline sample hold er (Fig. 1). Attachment of the
specimen to the support i s usually done with nail polish for
lighter, coalified mesofossils or with wax for heavier, permin-
eralized specimens. Other adhesive typically used for attaching
specimens to SEM stubs can also be used. Removal of
specimens attached with nail polish can be done easily with a
thin blade of a knife, particularly if the contact to the adhesive is
small.
The specimens are usually studied also by SEM either prior to
or after SXRTM. No additional sample preparation is necessary
for investigation of specimens that have previously been coated
with gold/platinum and analyzed with SEM. The coating does
not obscure internal features. Remounting of spe cimens from
SEM stubs may facilitate reconstruction, but is not a
requirement sinc e re-orientation of spec imens that have be en
mounted obliquely can be achieved physically using a
goniometer (Mader et al., 2011) or digitally using the
reconstruction software (Fig. 2).
Initial examina tion uses 2-D r adiographs taken for different
sample orientations using a parallel beam. Photons transmitted
through the sample are converted to visible light by a scintillator
screen. The image can then be magnified using light microscope
optics before being recorded by a digital camera (Fig. 2.1, 2.2).
Data from 2-D radiographs provide useful but cumulative
FIGURE 1—Close-up view of the TOMCAT end station showing the sample
holder with a fossil flower mounted on a brass stub, 3 mm in diameter, in front
of the microscope. (Paul Scherrer Institute photo).
FRIIS ET AL.—3-D VISUALIZATION OF PLANTS USING SRXTM 685
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information on the internal structure of the specimen along the
beam path. However, it is not possible to determine from 2-D
projections if a specific fe ature is loca ted at the front or back of
the specimen. To access such 3-D information, a second step
requires combining the recor ded radiographic dataset (reo rdered
into sinograms) using algorithms ba sed on Fourier analysis (e.g.,
filtered back-projection) or iterative methods.
SRXTM takes advantage of the absorption and refraction of
X-rays during their i ntera ct ion with matter resulting in different
imaging modalities. In absorption contrast tom ographic m icros-
copy, X-rays are selectively a ttenuated as they traverse the
sample according to the Bee r-Lambe rt law:
I ðZ; EÞ¼I
0
ðEÞexp
R
l
l
ðZ; EÞdz
;
where I(Z,E)andI
0
(E) are the X-ray beam intensity after and
before the specimen. The linear attenuation coefficient l
l
(Z,E)
strongly depend s on the atomic number Z: this provides high
contrast between materials with different densities, if the X -ray
beam energy E is properly selected.
When the ana lyzed sample is made of light elements, or
elements with a similar atomic number Z, contrast i s i nstead
obtained by exploiting the refractionoftheX-raybeamat
material boundaries in the study object and the resulting
interference phenomena. The refraction angles are small, but
can be determined with great accuracy using phase contrast
techniques. T he methods c ommonly deployed in paleontology
use the free-spac e propagation approach (Snigirev et al., 1995;
Cloetens et al., 1996). The main adva ntages with respect to
other existing approaches, such as interferometry (Bonse an d
Hart, 1965; Weitkamp et al., 2005) and analyz er syste ms (Davis
et al., 1995; Chapman et al., 1997) are the high spatial resolution
and uncomplicated setup that d oes not require additiona l
hardware. In the simplest case, pure, so-called, edge-enhance-
ment is exploited by increasing the distance between the sample
and the detector. Fresnel fringes are localized at domain
boundaries and arise from th e interference of the refracted and
the directly transmitted beam in case of spatially (partially)
coherent r adiation. This permits clear visualization of internal
boundaries, although the actual contrast between regions with
different composition is not improved compared to standar d
absorption contr ast tomography. This technique is routinely
used for understanding the internal struc tu re of fossil flowers,
fruits, seeds, and other mesofossils.
FIGURE 2—SRXTM images of holotype of Silvianthemum suecicum (S100376) from the Late Cretaceous
˚
Asen locality, Sweden; dataset acquired using a 103
objective and 20 lm thick LAG:Ce scintillator (voxel size 0.74) at 10 keV; specimen charcoalified and mounted obliquely on SEM stub. 1, 2, 2-D dark and flat
field corrected radiographic projections for two different sample orientations; 3, 2-D orthoslice of flower bud in longitudinal view through one style, sample not
re-oriented; 4, 5, 3-D surface rendering showing external morphology of flower bud in apical and lateral views, respectively; 6, electronically re-oriented
longitudinal section through center of flower bud in 3-D cut voltex (transparent rendering between orthoslices 740–780). Scale bars¼500 lm.
686 JOURNAL OF PALEONTOLOGY, V. 88, NO. 4, 2014
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More advanced approaches enable the extra ction of (phase)
information coded in the observed Fresnel fringes to further
boost the contrast between domains that have similar compo-
sitions. The simplest algorithms for phase retrieval (Bronnikov,
2002; Paganin et al., 2002; Groso et al., 2006) work with data
acquired at one single sa mple-de tector distance . In this case the
setup and protocol is the same a s for standard absorption based
experiments. These technique s are not fully quantitative and
rely on different assumptions, whic h ca n often be partially
relaxed, but the incre ased contrast is generally helpful in
particularly low absorbing specimens. The concomitant reduc-
tion in spatial resolution, which is sometimes observed, can be
mitigated by including the high frequenc y component of the
original data.
Fully quantitative, high resolution results (holotomography;
Cloetens et al., 1999) can be obtained using datasets a cquired at
multiple sample-detector distances. Holotomography is, how-
ever, rarely use d bec ause of the m ore complicated setup, slower
data acquisition and more complex data post-processing tha t is
required.
RECENT DEVELOPMENTS IN SRXTM TECHNIQUES
Spatial resolution.—For parallel beam geometry, the available
field of view is determined by the optical configuration chosen
and the resolution that is required. In general, the large field of
view necessary to accommodate a sample of large volume results
in a low magnification dataset whereas higher resolution can be
achieved if the field of view is more restricted. For instance, a
203 objective coupled to a sCMOS (scientific complementary
metal-oxide-semiconductor) detector provides a pixel size of
0.375 lm across a field of view of 0.8530.7 mm
2
, which is often
insufficient for the complete observation of larger specimens at
this resolution. When expansion of the available field of view in
the direction parallel to the rotation axis is desirable, but high
resolution is still required, a stack of several independent
tomographic scans can be acquired. However, an increase of
the field of view in the direction perpendicular to the rotation axis
cannot be obtained by simply juxtaposing single tomographic
datasets in this way. Instead it is necessary to merge projections
covering the desired field of view laterally prior to tomographic
reconstruction.
Although a multi-fold lateral expansion is technically feasible
(Haberthu¨r et al., 2010), for most of the mesofossil material
examined so far at the SLS a two-fold extension is often
sufficient. In such cases, prior to the scan, the rotation axis is
positioned at either side of the field of view, rather than in the
center, though still ensuring a small overlap for 1808 opposed
projections. Then, equiangular distributed projections over 3608
are acquired. The total number of projections is also increased
compared to standard tomographic scans, so as to still satisfy the
sampling theorem. Subsequently, projections acquired at an angle
h8 and h8þ1808 need to be merged, for instance using the
overlapping region and a cross-correlation technique, if the exact
position of the rotation axis is not known a priori. A single
tomographic volume can then be obtained using the merged
projections and a standard reconstruction algorithm.
In studies performed so far, high resolution imaging of small
fossil flowers with a diameter smaller than 0.7 mm has mostly
been accomplished using a 203 objective and a 20 lm thick
LAG:Ce (Cerium doped Lutetium Aluminum Garnet) scintillator
screen. Specimens as small as 0.3–0.4 mm, as is the case for
instance for megaspores, would entirely fit in the field of a 403
objective and could potentially be resolved even more finely
providing that the experimental setup is optimized for this
purpose. If higher magnification microsc ope objectives are
coupled to thinner scintillator screens, spatial resolution can be
pushed to the sub-micron regime even for experimental setups in
parallel beam geometry as is shown here for the Cretaceous
megaspore Arcellites (Fig. 3). The improvement in resolution
provided by this configuration is evident (Fig. 3.3). The 2-D slice
(Fig. 3.1) was extracted from a tomographic dataset acquired
using a 203 objective coupled to a 20 lm thick LAG:Ce
scintillator. Comparison with the slices (Fig. 3.2, 3.3) originating
from a tomographic volume obtained with a 403 objective and a
5.9 lm thick LSO:Tb (Terbium doped Lutetium Oxyorthosilicate,
Lu
2
SiO
5
) scintillator screen shows clearly the details of the
megaspore wall structure, including an outer layer penetrated by
narrow, straight canals about 0.4 lm in diameter and 7 lm long.
Three-dimensional reconstructions show the spatial distribution
of the canals (Fig. 3.4–3.6). Based on these results a new species
of Arcellites will be established and a detailed description of
megaspore morphology and wall structure presented (Friis et al.,
2014b). Ongoing optimization of the setup used for the initial
acquisition of the data (Fig. 3.2, 3.3) includes improvement of the
scintillator screen positioning system, ensuring homogeneous
focus across the entire field of view, and a pre-processing
alignment step to correct for any possible mechanical vibrations
or imperfections in the rotation that would lower resolution.
Density resolution.—During the past few years, microtomo-
graphy end-stations at third generation synchrotron sources have
been optimized in important ways resulting in datasets of
astonishing quality. In early applications of SRXTM to coalified
Cretaceous mesofossils, phase retrieval approaches were often
necessary to achieve the desired resolution (Friis et al., 2007).
However, now, simple absorption contrast is often sufficient.
New developments in detector technology (e.g., sCMOS), as well
as improvements in scintillating materials, have significantly
improved the signal-to-noise ratio in tomographic datasets and
resulted in improved density resolution. These developments have
also enhanced the efficiency of the tomography setup, reducing
the time necessary for the acquisition of a high-resolution scan by
approximately a factor of 20. This improvement in efficiency
enables the acquisition protocol to be optimized (e.g., increased
number of projections, frame-averaging) to boost signal-to-noise
ratio and ultimately density resolution. When applied to these
highest quality tomographic datasets, new phase retrieval
algorithms permit density resolution to be pushed even further.
Efficient data acquisition and increased computing power also
allows the complementarity of absorption and phase contrast
tomographic microscopy (spatial resolving power vs. density
resolving power) to be easily exploited. A single specimen can be
readily examined using both absorption and phase contrast
tomographic microscopy. For small, low absorbing samples (1
mm in diameter or smaller), such as many Cretaceous flowers, it
is generally sufficient to acquire one tomographic dataset with the
object positioned as close to the microscope as mechanically
allowed by the setup (typically at least 5 mm). From these data
two different tomographic volumes can be reconstructed.
Projections can be reconstructed using algorithms for absorp-
tion-based tomography providing the 3-D distribution of the X-
ray linear attenuation coefficient with edge-enhancement. Despite
little contrast due to the small difference in the linear attenuation
coefficient between air and the specimen, datasets reconstructed
in this way are characterized by high resolution and sharp edges.
Alternatively, information coded in the Fresnel fringes in the
same projections can be unraveled by phase retrieval approaches
prior to tomographic reconstruction to yield the 3-D distribution
of the pseudo-phase information (compare Fig. 3.2, 3.3). In this
case the results are generally not truly quantitative, and phase
contrast tomographic volumes often have lower resolution, but
the boosted contrast is useful to differentiate among domains with
similar compositions. Both approaches have inherent
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