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Spectrochimica Acta Part B
High spatial resolution X-ray microdiffraction applied to biomaterial
studies and archeometryB
A. Cedolaa,*, S. Lagomarsinoa, V. Komlevb, F. Rustichellib, M. Mastrogiacomoc,
R. Canceddac, S. Militad, M. Burghammere
aIstituto di Fotonica e Nanotecnologie-CNR, V. Cineto Romano 42, Roma 00156, Italy e INFM, Unita’ di Ancona, ItalybIstituto di Scienze Fisiche, Universita di Ancona, Via Ranieri 65, Ancona I60131, Italy
cIstituto Nazionale per la Ricerca sul Cancro and Dipartimento di Oncologia Biologia e Genetica, Universita’ di Genova, Largo R. Benzi 10,
Genova 16132, ItalydIstituto per la Microelettronica e Microsistemi (IMM)-CNR, Sez. Bologna, via Gobetti 101, Bologna I-40129, Italy
eESRF, B.P. 220, Grenoble, Cedex F-38043, France
Received 1 November 2003; accepted 1 May 2004
Abstract
The high spatial resolution X-ray microdiffraction by using X-ray optics can provide unique information on regions with very high
gradients in physical quantities, as in the case of interfaces. Among the several available X-ray optics for synchrotron radiation producing
high intensity micron and sub-micron beams, the X-ray waveguide (WG) can provide the smallest X-ray beam in one direction. Moreover, its
applicability has been widened by an improved set-up installed at ID13 beamline at ESRF, where a new undulator is combined with an
horizontally focusing mirror. In this work, we show different applications of waveguide-based microdiffraction, the first two regard biological
problems and in particular the structural analysis of newly formed bone in ceramic scaffolds. The second application regards archeometry and
in particular the sulphatation process and the thin gypsum crust formation on the surface of carbonate rocks (travertine, marbles), due to the
exposure of the monuments at aggressive atmospheres. In the three cases, the local structural information derived thanks to the high spatial
resolution demonstrates the power of the microdiffraction technique based on WG, and the possibility to apply this new methododogy in
different scientific fields.
D 2004 Elsevier B.V. All rights reserved.
Keywords: Microdiffraction; X-ray waveguide; Orthopaedic prosthesis; Bone marrow stromal cells; Cultural heritage
1. Introduction
The high demand of thorough characterization of
materials and processes requires development of advanced
diagnostic methods. One of the important figures of merit in
many cases is the spatial resolution. The X-ray micro-
diffraction technique combines diffraction, which is a
powerful tool for structural analysis, with the high spatial
0584-8547/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.sab.2004.05.031
B This paper was presented at the International Congress on X-Ray
Optics and Microanalysis (ICXOM XVII), held in Chamonix, Mont Blanc,
France, 22-26 September 2003, and is published in the special issue of
Spectrochimica Acta Part B, dedicated to that conference.
* Corresponding author. Tel.: +39 641522271; fax: +39 641522220.
E-mail address: [email protected] (A. Cedola).
resolution. To this purpose, the X-ray beam must, in general,
be conditioned. In principle, a simple pinhole could do this
task, but photon flux would be lost. Therefore, focusing X-
ray optics must be employed to concentrate photon flux in
small dimensions, as lenses do in the visible spectrum.
Unfortunately, there are severe problems in fabricating
optical elements for hard X-rays capable to reach sub-
micrometer spatial resolution. The advent of the high
brilliant synchrotron radiation sources gave new impulse
to research for innovative X-ray optics. At present, the
available optics for hard X-rays are the Fresnel Zone Plates
[1] based on diffraction, the refractive lenses [2] based on
refraction, the capillaries [3] and curved mirrors [4] based
on total reflection and the X-ray waveguides (WGs) [5–7]
59 (2004) 1557–1564
Fig. 1. Schematic representation of the waveguide structure.
A. Cedola et al. / Spectrochimica Acta Part B 59 (2004) 1557–15641558
based on standing waves which have demonstrated the
capability to provide the highest spatial resolution (in one
dimension) up to now.
Since the initial experiments using the WG [5], remark-
able improvements have been done with respect to optics
efficiency [8], and with respect to integration of the optical
element in a microdiffraction set-up and to experimental
procedures [9,10]. In this work, we present three recent
applications of microdiffraction using the WG in the field of
biology and archeometry.
2. Experimental
2.1. X-ray waveguides
A typical waveguide structure (see Fig. 1) consists (from
bottom to top) of an ultra-flat substrate, a metal layer few
tens of nm thick, a guiding layer made of a low-density
material having a thickness of the order of 100 nm and a
metal cap layer a few nm thick. This structure allows the
formation of a strong X-ray standing wave (XSW) field
inside the guiding layer with the spatial periodicity depend-
ing on the incident angle. When the XSW periodicity is
equal to an integer fraction of the layer thickness, a strong
resonance takes place, with an enhancement of the electro-
magnetic (EM) field hundredfold with respect to the
incoming EM field intensity. As with mode excitation in
Fig. 2. Scheme of the set-up used for microdiff
microwave resonators, the resonantly excited EM field can
travel along the waveguide and exit at its end. Only when
the resonance takes place an appreciable intensity can be
measured at the exit of the waveguide. Several modes can
therefore be considered, but in the following, we will focus
our attention only on the first resonance mode.
2.2. Beam properties
The properties of the beam exiting the waveguide can be
summarized as follows (to simplify the discussion, we
consider a reference frame where the waveguide surface is
in the horizontal plane):
the beam is confined in the vertical direction, with a Full
Width at Half Maximum (FWHM) equal to one half the
guiding layer thickness; typical values of FWHM are of
the order of 40–50 nm;
the exiting beam is divergent with a divergence of
typically 1 mrad (cwavelength/guiding layer thickness);
the beam is highly coherent and can be approximated in
the vertical direction by a gaussian beam, in strict
analogy with a laser beam;
in the horizontal direction, the beam remains unaltered;
if the incident beam is a plane wave, then the beam
exiting the waveguide is a cylindrical wave;
the gain (defined as the ratio between the flux density at
the exit divided by the incoming flux density) has reached
values of about one hundred in recent experiments, with an
improvement of three orders of magnitude during the last 3
years [8]. Gain can be also understood as the increase in
flux with respect to an hypothetical slit having an aperture
equal to the FWHM guiding layer thickness value.
2.3. X-ray microdiffraction set-up
The WG is mounted on an X-ray scanning microscope
(Fig. 2) for local structural analyses with sub-micron
raction experiment based on waveguide.
A. Cedola et al. / Spectrochimica Acta Part B 59 (2004) 1557–1564 1559
resolution installed at the microfocus beamline ID13 at the
European Synchrotron Radiation Facility (ESRF) in Gre-
noble [11]. As shown in Fig. 2, this microscope combines
the standard diffraction set-up with a focusing device. After
a preliminary definition by a slit system, the monochromatic
(k=0.9755 2) synchrotron radiation is horizontally focused
to about 3 Am by a laterally graded multilayer bent mirror
[12]. The beam emerging from the mirror impinges on the
waveguide which maintains the horizontal properties of the
beam and focuses it vertically to a dimension that in this
specific case is 0.05 Am (Full Width at Half Maximum) with
a divergence of about 1 mrad. The sample is adjusted at a
distance of 200 Am from the waveguide exit so that the spot-
size on the sample is about (3�0.3) Am2. A piezo-scanning
stage with 0.1 Am repeatability allows the sample to be
vertically scanned through the sub-micro beam. The flux
provided at the sample position is about 1010 ph/s and a flux
density of about 1016 ph/s/mm2.
The diffraction pattern of the sample is recorded by a
MAR CCD detector with an entrance window of 130 mm
diameter and pixel size of (64.45�64.45) Am2. The typical
exposure time for each image is 10 s.
Two optical microscopes looking at the sample from top
and from a side, allow the sample alignment and the
monitoring of the small investigated sample region.
Fig. 3. Electron microscopy image of sample A.
3. Results
3.1. Newly formed bone at prosthesis interface
Events leading to the integration of an implant into a
bone, determining the performance of the device, take place
largely at the tissue/implant interface [10–12]. After
implantation, reactions occur at the tissue/implant interface
that lead to time-dependent changes in the tissues and in the
surface characteristics of the implant material.
Up to now, the details of the interactions between tissue
and implant are still poorly understood being a complex
problem. In particular, as it is well known, the inorganic
component of the mature bone is described as highly
substituted hydroxyapatite (HA), but there is a long-stand-
ing controversy regarding the nature of the earliest state of
the bone. Two primary candidates for new bone mineral are
amorphous calcium phosphate (ACP) and octocalcium
phosphate (OCP), which are unstable and rapidly convert
to HA [13].
The major goal of the following experiment using the set-
up of Fig. 2 is to clarify the processes involved in bone
formation at the interface with the implant. In particular, the
study involves two different samples with different sub-
strates, a Yttria-stabilized Tetragonal Zirconia Polycrystal
(Y-TZP) coated with bioactive glass (RKKP bioglazeR)(sample A) and Y-TZP uncoated device (sample B). The
samples were obtained from highly pure, medical grade
powders (Yttria-stabilised ZrO2, Y-PSZ stabilised with 3
mol% Y2O3, or 5.5wt.%; Tosoh TZ3YB, Japan). Cylindrical
rods (4 mm in length, 2 mm in diameter) were prepared by
extrusion of a suitable paste made of plastifiers and flowing
agents that incorporate ZrO2 powders (about 90%). After
drying in normal atmosphere, all green ZrO2 substrates
obtained, were fired in a laboratory kiln with the following
thermal cycle: increase at the rate of 100 8C h�1 up to a final
temperature of 1550 8C, steady temperature for 1 h and
cooling at the rate of 200 8C h�1. After firing, samples were
refined. Rods were first cut with a diamond saw and then
their ends were rectified. The consistency of the ZrO2
ceramic bodies obtained, proved to be compact, without
apparent porosity and with homogeneous surfaces (no
asperity).
The samples were manufactured and implanted in the
condyle of rat femur under general anaesthesia. The details
of ceramics preparation and coating of samples are
described elsewhere [14]. Thirty days after implantation
the rat was sacrificed and samples A and B were obtained
cutting the femur perpendicular to the axis of the implant
and treated to obtain a thin section. Previous work dealt with
in vivo results [15] (carried out strictly following the
European and Italian Laws on animal experimentation)
and with morphological studies obtained with electron
microscopy. The same sample regions examined by electron
microscopy (see Fig. 3) have been accurately scanned
through the micro-beam of the WG and diffraction patterns
have been acquired by the CCD.
The measured diffraction spectrum of the reconstructed
bone at the interface region of sample A is compared in Fig.
4 with the spectrum of the cortical native bone. Spectra from
sample B in corresponding regions are quite similar to
sample A spectra. The analysis of the spectra reveals a
marked difference between the newly formed and the native
bone. The spectrum of the latter is perfectly compatible with
the hexagonal system of the HA, as well known in literature
[16], while the spectrum of the new bone, both for sample A
and sample B, appears to be quite different. Careful analysis
carried out comparing the ASTM Standard Diffraction
Fig. 5. Intensity variation of the OCP reflections (closed dots) and the
Fig. 4. Comparison of the experimental spectrum of native cortical bone
(straight line) with the spectrum of the newly formed bone (line with open
dots).
A. Cedola et al. / Spectrochimica Acta Part B 59 (2004) 1557–15641560
Tables revealed that the main reflections are compatible
with the presence of octocalcium phosphate (OCP) which
can be substantial in the first step of bone formation [17].
Nevertheless, also in the new bone spectrum the 002
reflection from HA is present.
The power of this micro-diffraction technique can be
clearly seen in Fig. 5 where the intensity variation of the
reflections [130]+[112] of OCP at d=3.1454 2 and [002] of
HA at d=3.4275 2 is studied at the interface region. The
high spatial resolution reveals the abrupt transition from
OCP to HA at the interface of the sample B (Fig. 5b) while a
smoother behaviour is clear for sample A (Fig. 5a). Indeed,
the presence of HA in sample A is extended in a deeper
region with respect to sample B and the thickness of the
zone at the interface where both phases coexist is about 3
and 60 Am for sample B and A, respectively. These results
HA reflections (open dots) for sample A (a) and sample B (b).
A. Cedola et al. / Spectrochimica Acta Part B 59 (2004) 1557–1564 1561
suggest the effectiveness of the coating process for the
osteointegration mechanism which is accelerated by the
bioactive glass.
3.2. Newly formed bone induced by marrow stromal cells
A recent successful experiment has been performed to
analyse structurally the newly formed bone induced by bone
marrow stromal cells loaded on a bioceramic scaffold and
implanted in a mouse model. In particular, in order to study
the bone growth mechanism, the interface between the new
bone and the scaffold (see Fig. 6b) has been analysed with
the high spatial resolution set-up of Fig. 2.
Fig. 6a shows the different patterns collected in the
region where the bone has grown (region A), in the region
where a fibrous tissue is present (region B) and in the
scaffold substrate (region C). The three patterns appear
much different. In particular in region A the typical ring
distribution of the HA represents the mineral particles of the
newly formed bone. In region B, the diffuse scattering
shows the amorphous state of the fibrous tissue where the
mineral particles are not yet formed. This phase is not
studied in details. In region C, the pattern of the scaffold
appears as rings composed of spikes. This is due to the large
grain dimension which is comparable to the beam size, of
the order of few microns.
Fig. 6. Diffraction patterns (a) collected in the three regions indicated in the optica
marked region represents the analyzed region.
The whole region marked in Fig. 6b has been scanned
with the micro-beam of the WG and different patterns have
been collected in order to provide information on the bone
growth with respect to the scaffold geometry. A detailed
analysis of both Wide Angle X-ray Scattering (WAXS) and
Small Angle X-ray Scattering (SAXS) patterns has been
carried out (see Fig. 7). Fig. 7a represents the WAXS
pattern collected in region A. Fig. 7b is a zoom of the
central region of Fig. 7a and it presents an anisotropic
SAXS pattern which shows that the mineral particles of
the bone tissue have elongated shape and are non
randomly distributed. Fig. 7c and d shows respectively
the radial distribution of the 002 reflection obtained from
the WAXS pattern and the radial distribution of the SAXS
pattern. Fig. 8a and b represents the azimuthal intensity
distribution of respectively the 002 reflection of the pattern
7a and the SAXS of Fig. 7b. The SAXS intensity exhibits
a minimum where the WAXD intensity is maximal and
vice versa. This means that the mean orientation of the
crystallographic c-axis corresponds exactly to the longest
axis of the mineral particles and therefore the bone mineral
particles grow in the direction of the c-axis. This relation
has been found for all the examined positions. Careful
analysis is being carried out on the diffraction pattern in
different locations in order to asses the growth mechanism
of bone in relation to the scaffold.
l microscope image in (b). The three regions are explained in the text. The
Fig. 7. (a) AWAXS pattern collected in region A of Fig. 6. (b) A zoom of the central region of (a), and it presents the SAXS pattern. (c and d) Radial intensity
distribution of respectively the 002 reflection of pattern (a) and the SAXS of (b).
A. Cedola et al. / Spectrochimica Acta Part B 59 (2004) 1557–15641562
3.3. Application in archeometry
There is an extensive literature about the deterioration
mechanisms of natural building stones [18–23]. Acidity in
the air is essentially caused by pollutants, such as sulfur and
nitrogen oxides, which are emitted into the atmosphere by
sources related to industry, transportation and heating.
These species are transformed, through complex reaction
Fig. 8. Azimuthal intensity distribution of the 002 reflect
pathway, into gaseous nitric and nitrous acids and into
acidic sulfates as suspended particles. Although in recent
years, we have assisted to a decay of the levels of pollution
in the urban areas in Europe, we have still consistent levels
of HNO3 and other aggressive species as sulfur dioxide and
ozone. As well-known, SO2 and NOx react with calcium
carbonate rocks to form sulfates and nitrates, which, due to
their solubility in water, may be drained away or, if
ion (a) of pattern 7a and the SAXS of Fig. 7b (b).
A. Cedola et al. / Spectrochimica Acta Part B 59 (2004) 1557–1564 1563
protected from the rain, may form crusts that eventually
exfoliate.
In Ref. [23], it was proposed a quantitative mathematical
model to describe the time evolution of this process and a
numerical approximation was performed to show the
qualitative behaviour of the solutions. This could be very
useful in preventing further damage of building stones. In
fact, experiences with protective indicate that we have to be
very careful in using such kind of products to protect our
monuments. Effective simulation tools could be useful to try
to assign a degree of priority for the cleaning of the different
monuments, also considering the local geometry and the
exposure of the concerned rock.
In order to validate the mathematical model, a high
spatial resolution is needed to study the sulfatation process
at the interface between the crust and the substrate. For
this purpose, a feasibility experiment has been carried out
on a thin section of a sample from Teatro Marcello of
Fig. 9. (a and b) Diffraction patterns recorded from two different depth po
Rome using the set-up of Fig. 2. Diffraction patterns
recorded from two different depth positions (Fig. 9a and
b) allow one to determine the difference in structure
(crystal lattice spaces) and morphology (crystallite dimen-
sions) of the two diffracting regions, indicating the
possibility to follow the CaSO4 concentration profiles as
indicated in Fig. 9c. To quantify the data a proper
calibration using standard samples (Al2O3) has been
previously performed.
The main aim of this particular experiment was to check
the possibility to study the coexistence of CaSO4 and
CaCO3 using the WG set-up. Even if the sample analysed in
this particular experiment showed an interface region quite
large (160 Am), the final goal is to use the same approach to
study very abrupt interface as it is the case of samples
exposed at aggressive atmospheres in laboratory. In this
way, the sulfatation process can be studied for different
materials and different atmosphere conditions.
sitions, along the scan direction. (c) CaSO4 concentration profiles.
A. Cedola et al. / Spectrochimica Acta Part B 59 (2004) 1557–15641564
4. Conclusions
In this work, the power of the microdiffraction technique
using the waveguide has been shown presenting three
examples carried out at the ID13 beamline of ESRF
regarding different scientific fields. Mainly in the cases of
bone formation, the high spatial resolution provides unique
information on regions with very high gradients in physical
quantities, as in the case of interfaces.
In particular, the first two examples regarded the local
structural analysis of the newly formed bone and the
information which are derived provide an advance in the
understanding of bone growth. The last experiment regard-
ing cultural heritage demonstrates the possibility to use this
advanced technique to study the sulfatation problem which
forms thin crust on the deteriorated monuments.
Acknowledgement
It is a pleasure to thank Dr. N.N. Aldini and Prof. R.
Giardino who provide the thin sections of the samples with
the coated and uncoated orthopaedic devices.
This work was partially supported by the program PURS
of the National Institute for the Physics of Matter (INFM).
References
[1] E. Anderson, D. Kern, in: A.G. Michette, G.R. Morrison, C.J. Buckley
(Eds.), X-Ray Microscopy, Spring, Berlin, 1990, pp. 75–79.
[2] A. Snigirev, V. Kohn, I. Snigireva, B. Lengeler, Nature 384 (1996)
49–51.
[3] D.J. Thiel, D.H. Bilderback, A. Lewis, E.A. Stern, Nucl. Instrum.
Methods, A 317 (1992) 597–600.
[4] P. Kirkpatrick, A.V. Baez, J. Opt. Soc. Am. 38 (1948) 766–774.
[5] S. Lagomarsino, W. Jark, S. Di Fonzo, A. Cedola, B. Muller,
P. Engstrom, C. Riekel, J. Appl. Phys. 79 (1996) 4471–4473.
[6] Y.P. Feng, S.K. Sinha, E.E. Fullerton, G. Grubel, D. Abemathy,
D.P. Siddons, J.B. Hastings, Appl. Phys. Lett. 67 (1995) 24–26.
[7] W. Jark, S. Di Fonzo, S. Lagomarsino, A. Cedola, E. Di Fabrizio,
A. Brahm, C. Riekel, J. Appl. Phys. 80 (1996) 4831–4836.
[8] W. Jark, A. Cedola, S. Di Fonzo, M. Fiordelisi, S. Lagomarsino,
N.V. Kovalenko, V.A. Chernov, Appl. Phys. Lett. 78 (2001)
1192–1194.
[9] S. Di Fonzo, W. Jark, S. Lagomarsino, C. Giannini, L. De Caro,
A. Cedola, M. Muller, Nature 403 (2000) 638–640.
[10] A. Cedola, V. Stanic, M. Burghammer, S. Lagomarsino, F. Rustichelli,
R. Giardino, N. Nicoli Aldini, M. Fini, V. Komlev, S. Di Fonzo, Phys.
Med. Biol. 48 (2003) N37–N48.
[11] M.Muller, M. Burghammer, D. Flot, C. Riekel, C.Morawe, B.Murphy,
A. Cedola, J. Appl. Crystallogr. 33 (2000) 1231–1240.
[12] P. Ducheyne, Q. Qiu, Biomaterials 20 (1999) 2287–2303.
[13] S.V. Dorozhkin, M. Epple, Angew. Chem. Int. Ed. 41 (2002)
3130–3146.
[14] A. Krajewsky, A. Ravaglioli, M. Mazzocchi, M. Fini, J. Mater. Sci.,
Mater. Med. 9 (1998) 309–316.
[15] N. Nicoli Aldini, M. Fini, G. Giavaresi, P. Torricelli, L. Martini,
R. Giardino, A. Krajewski, A. Ravaglioli, M. Mazzocchi, B. Dubini,
M.G. Ponzi Bossi, F. Rustichelli, V. Stanic, J. Biomed. Mater. Res. 61
(2002) 282–289.
[16] H. Aoki, Science and Medical Application of Hydroxyapatite, JAAS,
Tokyo, 1991, p. 179.
[17] M. Mathew, S. Takagi, J. Res. Natl. Inst. Stand. Technol. 106 (2001)
1035–1044.
[18] F.H. Haynie, Durab. Build. Mater. 1 (1982/83) 241–254.
[19] S. Tambe, K.L. Gauri, S. Li, W.G. Cobourn, Environ. Sci. Technol. 25
(1991) 2071–2075.
[20] K.L. Gauri, R. Popli, A.C. Sarma, Durab. Build. Mater. 1 (1982/83)
209–216.
[21] K.L. Gauri, J.A. Gwinn, Durab. Build. Mater. 1 (1982/83) 217–223.
[22] F. Garbassi, E. Mello, M. Laurenzi Tabasso, Durab. Build. Mater. 3
(1985) 51–58.
[23] D. Aregba-Driollet, F. Diele, F. Guarguaglini, R. Natalini, preprint
IAC-CNR (2001).