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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: cedola@ifn.cnr.it (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).

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