E Mccarthy

R T Rajendra Kumar

B Doggett

S Chakrabarti

R J O'haire

S B Newcomb

J.-P Mosnier

M O Henry

E Mcglynn
email: enda.mcglynn@dcu.ie.

Effects of the crystallite mosaic spread on integrated peak intensities in 2θ-ω measurements of highly crystallographically textured ZnO thin films

We report x-ray diffraction (2θ-ω and rocking curve) and transmission electron microscopy measurements on crystallographically textured ZnO thin films of varying thicknesses and crystallite mosaic spread deposited by pulsed laser deposition on Si. The integrated areas of the (0002) ZnO reflections in 2θ-ω mode do not scale with film thickness and in some cases show discrepancies of two orders of magnitude compared to expectations based solely on sample thicknesses. Intensity differences of this type are regularly used in the literature as indications of differences in sample crystallinity or crystal quality. However transmission electron microscopy data of our samples show no evidence of amorphous deposits or significantly varying crystal quality in different films. X-ray rocking curves of these samples do show substantial variations in the mosaic spread of crystallites in the ZnO films which are the origin of the differences in integrated

areas of the (0002) ZnO reflections in 2θ-ω measurements. We outline a generally applicable model to treat the 2θ-ω mode peak intensities which shows good agreement with the experimental data (to within an order of magnitude) and which is much simpler than utilizing a full reciprocal space map approach to understand the x-ray diffraction data. We conclude that the normalized integrated intensity of the (0002) ZnO reflection in highly crystallographically textured ZnO thin films is strongly dependent on the rocking curve width in addition to the film thickness and the use of such intensities in isolation as measures of the thin film crystallinity or crystal quality, without reference to the rocking curve width, is likely to be misleading when making judgments of such aspects of the thin film structure.

Introduction:

ZnO thin films have been the topic of extensive research in the past decade, with the aim of using the attractive photonic properties of the material in photonic devices such as LEDs and laser diodes [1]. The ubiquitous tendency of nominally undoped ZnO towards n-type conductivity has meant that reliable and reproducible p-type doping at carrier and mobility levels required for device operation has been extremely difficult to attain.

Nevertheless, significant advances have been made in the quality of thin films grown by a number of techniques, including metal organic vapour phase deposition (MOCVD), molecular beam epitaxy (MBE) and pulsed laser deposition (PLD) [2]. In the majority of cases, the substrates used for ZnO thin film growth are either Si or one of the main sapphire planes (c-, a-, mor r-planes). The growth is in the form of a columnar grain formation along the ZnO c-axis with the c-axis preferentially oriented normal to the substrate surface (i.e. showing crystallographic texture). This growth mode is reported even on amorphous substrates such as glass [3] and appears to be related both to the high basal plane surface energy of ZnO and also proximity effects of neighbouring columnar crystallites, which will tend to increase the c-axis crystallographic texture as the layer thickness increases, to reduce strain effects associated with the interaction / coalescence of non-normal columnar crystallites [4,5]. The crystallographic texture is distinct from any surface texture (morphology) properties of the samples. We use the term "texture" solely in its crystallographic sense, in terms of preferred crystallite orientation.

X-ray diffraction (XRD) is an extremely useful workhorse technique in determining film

parameters including strain, crystallinity/crystal quality, texture, coherence length and inplane ordering [START_REF] Cullity | Elements of X-Ray Diffraction[END_REF]. A number of authors have recently used the peak intensity or integrated area of the (0002) ZnO reflection (or other reflections) as measures of the ZnO thin film crystallinity and/or crystal quality [START_REF] Fang | [END_REF][8][9][10][11]. In this paper we report x-ray diffraction (2θ-ω and rocking curve) data on highly textured ZnO thin films of varying thicknesses deposited by PLD on Si, which show only ZnO (0002) and, occasionally, (0004) reflections (in addition to substrate peaks). The integrated areas of the (0002) ZnO reflections in 2θ-ω mode do not scale with film thickness and in some cases show discrepancies of two orders of magnitude compared to expectations based on sample thicknesses and could potentially be attributed to differences in sample crystallinity or crystal quality. However, transmission electron microscopy (TEM) data on our samples show no evidence of amorphous deposits or significant variations in crystal quality. We treat the problem in terms of the effects of mosaic spread on the reciprocal lattice spots, revealed by x-ray rocking curve data, and outline the effect of such mosaic spreads on the 2θ-ω mode peak intensities. We conclude by discussing the absolute necessity to account for rocking curve widths in discussions of the normalized integrated intensities of the (0002) ZnO reflection in highly textured ZnO thin films, especially when considering aspects such as thin film crystallinity or crystal quality.

Experimental Conditions:

The growth apparatus has been described in detail elsewhere [12,13]. To summarise, ZnO thin films were deposited on Si (001) substrates using PLD by ablating a ZnO target (99.999%) in an oxygen atmosphere (99.999%, 75 mTorr) with a frequency quadrupled Nd:YAG laser (266 nm, 1.4 J/cm 2 , 6 ns pulse duration, repetition rate 10 Hz). The Si substrates were degreased prior to growth and preheated in-situ at 950°C for 5 minutes.

No effort was made to strip the native oxide. ZnO thin films with thicknesses in the range 30 -1000 nm (determined by spectroscopic ellipsometry and corroborated by TEM measurements on certain samples) were then deposited, maintaining the substrates at 300° C and the ZnO films were then annealed in-situ at 700°C for 5 minutes. Different samples were grown at times many months apart. For spectroscopic ellipsometry data analysis following measurement either a two layer or a three layer stack model was used to achieve a best fit to the data. In the former case, a measurement of the bare silicon substrate with oxide was made and used as the substrate layer in the model, removing the need to add an oxide layer to the model. In this case ZnO was the second layer, with a variable thickness. In the latter case, a Si substrate with a silicon dioxide layer and a ZnO layer was used with variable thickness. A Forouhi & Bloomer dispersion formula was used for the ZnO layer with 2 oscillators and 8 parameters (and using standard parameter values for the ZnO layer). Roughness layers were tried in the model for a number of samples; however they were found to not improve the fit and typically returned thickness values of zero. Thickness values obtained from spectroscopic ellipsometry were compared against values obtained from surface profilometry measurements across step edges (using substrates where one half of the deposition region was masked during the PLD deposition) for a number of test samples and in all cases excellent agreement was found. Furthermore, the agreement between thickness values from spectroscopic ellipsometry and cross-sectional TEM was excellent in all cases where comparisons were made.

The crystal structure was characterized by XRD (Bruker AXS D8 advance texture diffractometer). The XRD data in 2θ-ω mode were collected after careful optimization of the ω, φ and χ angles on the Si (004) reflection, to ensure comparability of the relative intensities (relative to the Si (004) reflection) of the ZnO (0002) reflection from sample to sample. The φ angle has been adjusted in some cases to reduce or eliminate the kinematically forbidden Si (002) reflection which is seen through double diffraction effects [14]. Rocking curve data were also collected for these samples (in addition to a limited number of φ scan measurements). Because of the extended period over which samples were grown significant variations in diffractometer x-ray output intensity were observed, so in all cases where comparisons are being made we use only (integrated) intensities suitably normalized to the Si substrate peak integrated intensity. Samples for TEM characterisation were thinned to electron transparency using standard focused ion beam milling procedures [15] and examined in a JEOL2000FX operating at 200kV.

Results and Discussion:

Figure 1(a) shows XRD diffractograms in 2θ-ω mode on four representative ZnO samples, i, ii, v and vi as listed in table 1 while figure 1(b) shows rocking curve data for the same samples. The rocking curves are fitted with a Gaussian lineshape, including a baseline offset, to determine the peak widths and the fit lines are shown in the figure. The small features marked "A" are due to the plastic-backed adhesive tape used to mount the samples, while the features marked "B" are due to K β radiation from the x-ray tube at ~ 62 0 and tungsten L α radiation from contamination of the x-ray tube at ~ 66 0 [16].

Changes in the central maximum position in figure 1(b) are due to (small) random sample tilts during the sample mounting procedure.

The 2θ-ω data show that the samples are highly textured ZnO, with significant contributions only from the ZnO (0002) planes (and occasionally the 2 nd order (0004))

and Si (004) planes. In one case (sample v) a small reflection due to the kinematically forbidden Si (002) reflection is seen also [14]. The samples show some evidence of slight strain, varying from sample to sample (and is least in thicker samples), but in all cases the measured strain is much less than 1%. The integrated intensities for all eight samples (after subtraction of background intensities) of the ZnO (0002) and Si (004) peaks are listed in table 1, in addition to the rocking curve full widths at half maximum (FWHM). The rocking curve widths were found to be independent of the sample φ angle in all cases. Table 1 also lists other key sample properties such as thickness (as determined by spectroscopic ellipsometry and corroborated for some samples by crosssection TEM data).

Table 1 (column 6, boldfaced) shows that the normalized integrated intensity (with respect to the Si (004) peak integrated intensity) per nm of the samples varies over a range of almost two orders of magnitude. As mentioned previously, a number of authors have recently used the peak intensity or integrated area of the (0002) ZnO reflection in isolation as measures of the thin film crystallinity [START_REF] Fang | [END_REF][8][9][10][11]. The results shown in table 1 based on the data in figure 1(a) would, if taken as a measure of film crystallinity, imply a significant difference in the crystalline to amorphous deposit ratio in our samples. However, the cross-sectional TEM data shown in figures 2 and 3 for the samples labelled (ii) and (v) in figure 1 and table 1, respectively reveal a crystallographically textured deposit in the form of columnar crystallites with their long axes lying mainly perpendicular to the substrate. The contrast variations seen in dark field micrographs (imaged using the (10-10) ZnO reflection) shown in figures 2(c) and 3 (c) are similarly fully consistent with the presence of a textured polycrystalline deposit and further emphasise that no amorphous oxide has been formed. These observations are similar to other reports for ZnO layer growth in Si substrates with a native oxide [3] and also confirm the spectroscopic ellipsometry measurements for both 40 nm and 425 nm layer deposits. XRD rocking curves shown in figure 1(b) however reveal substantial sample to sample variations in the degree of texture of the samples, with smaller rocking curve widths generally found in thicker samples, but with variations from this trend also present. The origin of this variation is due to (a) changes in film thickness, which lead, due to proximity effects of neighbouring crystallites, to changes in texture (generally smaller mosaic spread for thicker films) [5] and (b) small variations in growth parameters over the extended time period over which the sample set was grown, which are known to also affect sample texture [3].

The normalized (per unit volume of material) intensity of an XRD reflection in θ-2θ or 2θ-ω modes corresponding to a change in x-ray wavevector k is proportional to the absolute value squared of the Fourier transform of the x-ray scattering density with respect to k in the regime of kinematic diffraction appropriate for the thin, polycrystalline mosaic ZnO films studied here [START_REF] Cullity | Elements of X-Ray Diffraction[END_REF][START_REF] Als-Nielsen | Elements of Modern X-Ray Physics[END_REF]. For these samples the scattered intensity is spread over a range of k values centred on the reciprocal lattice points of the perfect single crystal because of (a) the effects of finite coherence length of the individual crystallites and (b) the mosaic spread of the crystallites [START_REF] Cullity | Elements of X-Ray Diffraction[END_REF]. The integral of the scattered intensity over this range in reciprocal space which gives a total intensity for reflections from this set of planes is proportional to the sample volume [START_REF] Cullity | Elements of X-Ray Diffraction[END_REF][START_REF] Als-Nielsen | Elements of Modern X-Ray Physics[END_REF][START_REF] Bauer | Optical Characterization of Epitaxial Semiconductor Layers[END_REF]. One can envisage this for the samples discussed here in the form of a density plot in k-space, with the density representing the normalized scattered intensity at that value of k, as shown schematically in figure 4. In figure 4 the shading of the (oblate spheroidally-shaped) reciprocal lattice spot indicates that this density and the size in various directions is a measure of the spread of the spot in reciprocal space (e.g. its FWHM). The spread of the spot in the direction normal to the sample surface (k ⊥ ) is determined by the out-of-plane coherence length of the crystallites (Scherrer formula [START_REF] Cullity | Elements of X-Ray Diffraction[END_REF]), while its extent in a direction parallel to the sample surface (k ⎪⎪ ) is predominantly determined in these samples by the mosaic spread of the crystallites (as measured by the rocking curve FWHM) [START_REF] Cullity | Elements of X-Ray Diffraction[END_REF][START_REF] Bauer | Optical Characterization of Epitaxial Semiconductor Layers[END_REF]. For a fixed thin film volume, the greater the mosaic spread the larger the volume of reciprocal space over which the scattered intensity is distributed and, since the total integrated density is proportional to the film volume, this implies that the peak density reduces at a rate inversely proportional to the square of the rocking curve FWHM (if, as in this case, it is independent of φ). The 2θ-ω mode of operation essentially scans through reciprocal space in a direction normal to the substrate surface (k ⊥ ) and thus measures the intensity along such a trajectory in k-space, while a rocking curve scans through reciprocal space in a direction parallel to the substrate surface (k ⎪⎪ ) [START_REF] Bauer | Optical Characterization of Epitaxial Semiconductor Layers[END_REF]. These points are shown schematically in figure 4 In Table 1 the product of the rocking curve FWHM squared multiplied by the integrated normalised intensity per nanometer oxide layer thickness is shown in column 8 (boldfaced and italicised), and it is seen that, in contrast to column 6, the values for all samples are of the same order of magnitude (with an average value of 2.21 x 10 -3 and a standard deviation of 0. 55 x 10 -3 ), which is consistent with the model outlined above (the sources of the remaining spread of values/discrepancies are briefly discussed below). It is clear that variations in rocking curve FWHM are responsible for substantial discrepancies (up to two orders of magnitude) in the normalized integrated intensity per nm of the (0002) peak for ZnO thin films and that these discrepancies are largely reconciled when variations in FWHM are taken into account with the model above. We note that the approach outlined above is suitable for analysis of highly textured thin films, such as those in our case which display a single lattice plane reflection (and higher orders of same). It will become less useful (and ultimately redundant) as the degree of texture reduces and different lattice plane reflections appear in the diffractogram (until finally the powder pattern is recovered when the texture vanishes).

The remaining spread in values in our data in column 8 of table 1 is primarily due to the practical difficulties encountered in normalizing with respect to the Si (004) peak integrated intensity. The sample orientation was optimized by adjusting the ω, φ and χ angles on the Si (004) reflection to achieve maximum intensity. However, the very low FWHM of this peak in angular terms (the rocking curve FWHM for Si (004) was measured as ~ 0.06 0 , data not shown) means that slight mis-adjustments due to the finite step size of the instrument and the sequential nature of angular optimizations can cause changes (~ 10's of %) in the Si (004) peak intensity in the 2θ-ω mode, which account for the majority of the remaining discrepancy in column 8. In fact, for samples measured in similar conditions on the same day on the same instrument (where the x-ray intensity is unlikely to change greatly) it is probably more accurate to use the unnormalised ZnO (0002) integrated intensity. However, when seeking to compare samples grown over extended time periods during which the x-ray source intensity may have changed (as in the present study), or where the measurement conditions have changed substantially, normalization with respect to the Si (004) integrated intensity is necessary.

Conclusions:

The normalized integrated intensity of the (0002) ZnO reflection in highly textured ZnO thin films is strongly dependent on the rocking curve FWHM in addition to the film thickness and the use of such intensities "in isolation" as measures of the thin film crystallinity or crystal quality, without reference to the rocking curve FWHM, is likely to be misleading in any assessment of these aspects of the thin film structure. We have outlined a model which shows that the product of the rocking curve FWHM squared and the integrated normalized intensity should be proportional to film thickness and this prediction compares very well with XRD data from a range of samples of varying thicknesses and degrees of mosaic spread deposited by pulsed laser deposition on Si. The remaining discrepancy is explained by the difficulty of normalizing to the very sharply defined Si (004) XRD peak. In recent times a number of reports on potential complexities and pitfalls in XRD characterization of thin film and nanostructured materials have appeared [14,[START_REF] Weidenthaler | [END_REF]. While the points discussed above have been recognised by the crystallography community for many years [20], they seem to be less well known among the thin film community and thus this note may be useful to re-emphasize them.

Acknowledgements:

Tables:

  , for examples of small (4(a)) and large (4(b)) mosaic spreads in a textured thin film of constant volume and constant out of plane coherence length (the shading of the ZnO (0002) reciprocal lattice spot on the left hand side indicates the normalized scattering density/intensity). Schematic representations (with indicative intensity and angular scales for comparative purposes) are shown for 2θ-ω and rocking curve scans of the two situations on the right hand side of figure 4. Hence the integrated intensity of the symmetric film reflections for a highly textured film in 2θ-ω mode should be proportional to the sample volume (film thickness) and inversely proportional to the rocking curve FWHM squared, or equivalently, the product of the rocking curve FWHM squared and the integrated normalized intensity should be proportional to film thickness. The use of integrated 2θ-ω intensities removes any variation due to changes in the out of plane coherence length from sample to sample.

Figure captions:Figure 1 :

 1 Figure captions:

Figure 2 :

 2 Figure 2: Cross-sectional TEM micrographs for the sample labelled (ii) in figure 1 and

Figure 3 :

 3 Figure 3: Cross-sectional TEM micrographs for the sample labelled (v) in figure 1 and

Figure 4 :

 4 Figure 4: Schematic representations of the ZnO (0002) reciprocal lattice spots (left side)

Figures:Figure 1

 1 Figures:

Figure 2 20 Figure 4 :

 2204 Figure 2: E. McCarthy et al

Table 1 :

 1 Summary of XRD data from samples studied.

	Sample	Thickness	Integrated	Integrated	Normalised	Normalised	FWHM	Normalised
	identifier	from	Intensity	Intensity	integrated	integrated	of	integrated
		spectroscopic	(peak area,	(peak area,	intensity of	intensity of	rocking	intensity of ZnO
		ellipsometry	arb. units)	arb. units)	ZnO (0002)	ZnO (0002)	curve on	(0002) peak per
		(nm; *			peak (arb.	peak per	ZnO	nm film
		indicates	ZnO	ZnO	units)	nm film	(0002)	thickness x
		corroborating	(0002)	Si(004)		thickness	peak	(FWHM of
		TEM	peak	peak		(arb. units)	(degrees)	rocking curve
		measurements)						on ZnO (0002)
								peak) 2
								(arb. units)
	i	33*	3.40E+01	1.48E+05	2.29E-04	6.95E-06	20	2.78E-03
	ii	40*	3.00E+01	1.13E+05	2.66E-04	6.64E-06	17.9	2.13E-03
	iii	87	1.81E+02	1.64E+05	1.10E-03	1.27E-05	15.35	2.99E-03
	iv	339	7.32E+02	1.74E+05	4.20E-03	1.24E-05	13.4	2.22E-03
	v	427*	7.77E+03	6.79E+04	1.14E-01	2.68E-04	2.3	1.42E-03
	vi	650	1.48E+03	1.13E+05	1.31E-02	2.01E-05	11.25	2.55E-03
	vii	761	3.26E+03	1.98E+05	1.65E-02	2.17E-05	8.5	1.57E-03
	viii	1057	6.85E+03	1.91E+05	3.58E-02	3.39E-05	7.75	2.04E-03

The authors gratefully acknowledge financial support from Science Foundation Ireland (Investigator Grant # 02/IN1/I95) and the Irish Higher Education Authority under the NDP. RTRK and EMcG also wish to acknowledge the International Visitors Programme of DCU which provided a fellowship for RTRK to visit DCU in the summer of 2010. We are grateful to Dr. Barry O'Connell and Mr. Henry Barry (DCU) for their assistance in performing ellipsometry measurements.


N K Manninen

R Escobar Galindo

N Benito

M Figueiredo

A Cavaleiro

C Palacio

S Carvalho

N M Figueiredo

Ag-Ti(C, N)-based coatings for biomedical applications: influence of silver content on the structural properties

Keywords: Sputtering, XPS, Raman, TEM

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Introduction

The increasing demand for sustainable products requires the development of new knowledge-based materials with advanced properties. These products are then expected to: last longer, have a better performance, be safe, and be more efficient. These requirements, which maybe seen as a rule of thumb for most known materials, gain particular importance when dealing with applications involving the human body: the socalled biomaterials [1]. Fatigue fracture and wear have been identified as major problems associated with implant loosening, stress-shielding and ultimate implant failure. Also, medical devices have sometimes to be the physically removed due to infection. In fact, these wear-related problems are being studied by the development of new functional biomaterials which can intend to have some improved physical, mechanical, tribological or biological properties [2][3][4]. The tendency in implant science is to replace the total hip prosthesis, which was usually made of a variety of materials such as metals, ceramics, polymers and composites, by those materials coated with protective thin-films.

Different types of PVD hard ceramic coatings were frequently considered as possible solutions to overcome the drawbacks mentioned above. In recent years, nanocomposite protective films have gained popularity due to their superior mechanical and tribological properties compared to traditional single-phase films. Superhard nanocomposites based on the combination of various transition metal nitrides, carbides and carbonitrides have been reported in last years [5][6][7][8]. Clearly control the nanoscale coating structure in order to achieved the best properties (enhanced hardness and tribological behavior) is the main motivation of these reports. Titanium carbonitride (TiCN) presents excellent wear resistance, high hardness and good corrosion resistance beyond its non-cytotoxic character. These properties justify that TiCN could be a potential coating for hip implants. TiCN has been studied in last decades specially the structural properties and their relation with the mechanical and tribological behavior, which determines the inservice performance in mechanical applications [3,4,9,10]. As example, Martínez-Martínez et al. reported that in TiCN coatings deposited by magnetron sputtering, a C enrichment led to the formation of an amorphous carbon phase that can help to decrease the friction coefficient and the wear rate, although promoting a hardness reduction, conditions giving the best performance in some applications, such as in hip implants [11].

Previous results obtained demonstrated that although these type of biomaterials presented good mechanical properties and low cytotoxicicty [3,12] they were prone to be colonized by Staphylococcus epidermidis. Hence this strengthens the urgent need of improving coatings incorporating antimicrobial properties. The current investigations supports that use of silver ion or metallic silver as well as silver nanoparticles can be exploited in medicine for burn treatment, dental materials, coatings stainless steel materials, textile fabrics, water treatment, sunscreen lotions and posses low toxicity to human cells [13,14]. Indeed, several studies have been performed describing the use of silver for biomedical applications [15][16][17]. However, they only consider silver antimicrobial action disregarding how silver is incorporated and/or its morphology. In fact the antiomicrobial property of silver is related to the amount of silver and the rate of silver released is correlated with structural properties.

The aim of the present study is the production and structural characterization of Ag-TiCN for hip implant applications and the focus is to study the influence of silver addition to the structure of TiCN coatings produced by DC reactive magnetron sputtering. According to the results obtained by several authors, the functional properties are strongly correlated with the coatings structure. On the one hand the mechanical/tribological behaviors are clearly governed by the balance between crystalline and amorphous phases. On the other hand, silver antimicrobial properties are mainly controlled by the amount of silver present in the system and by the surface to volume ratio, which depends on the morphology of silver clusters. Thus, in order to understand the functional properties of Ag-TiCN the knowledge of structure and morphology is primordial. In fact a deep investigation of the structure needs to be done first in order to understand the usually very complex results one obtains from tribological and biological tests. This work intends to contribute to this comprehension field.

Materials and Methods

Ag-TiCN samples were deposited by reactive DC magnetron sputtering from a highpurity Ti target (200 x 100 mm 2 ) onto polished and ultrasonically cleaned 316 L stainless steel (20 x 20 mm 2 ) and single crystalline silicon (100). High purity silver is incorporated on a second Ti target in the form of nuggets. Two series of samples were deposited using this Ti target with 6 and 14 Ag nuggets, resulting in relative Ag sputtering areas (only the preferential eroded zone of the target is considered) of 15% and 43%, respectively.

To further vary the Ag content in the films in both series the current density applied to each target was also changed. The substrates were previously sputter-etched for 20 minutes in an Ar + atmosphere. The depositions were carried out in Ar+C 2 H 2 +N 2 mixtures using an Alcatel SCM650 apparatus, with the substrates rotating at 70 mm over the target at a constant speed of 8 rpm. The base pressure in the deposition chamber was about 10 -3 Pa and rose up to values between 3.1 x10 -1 and 4.7x10 -1 Pa during the depositions. The films were grown at a constant temperature (573 K) and bias voltage (-70 V). Argon flow was kept constant at 60 sccm while the reactive gases fluxes (C 2 H 2 and N 2 ) were changed as it is indicated in Table 1. The variation on the reactive gases fluxes was performed in order to maintain approximately constant the C and the N contents, although the density current applied to the targets was changed. The stoichiometry of the deposited films was investigated by a Cameca SX 50 Electron Probe Microanalysis (EPMA) apparatus. Ball crater tests were used to measure the film thickness. The structure and phase distribution of the coatings were determined by Xray diffraction (XRD) using a conventional Philips PW 1710 diffractometer, operating with Cu K radiation in grazing incidence mode (angle of incidence of 1.5°).

The microstructure analysis was carried out on Tecnai G2 transmission electron microscope operated at 200 kV. The morphology of the coatings was evaluated by Scanning Electron Microscopy (EDAX-Nova nanoSEM 200).

In order to analyze the chemical bonding Raman Spectroscopy and X-ray Photoelectron Spectroscopy (XPS) were performed. Raman spectra were acquired with a Renishaw InVia Microscope. The excitation light was the 514.5 nm line of an Ar laser.

XPS spectra were acquired using a hemispherical analyzer (SPECS EA-10 Plus).The pass energy was 15 eV giving a constant resolution of 0.9 eV. The Ag 3d5/2 line at 367.9 eV was used in order to calibrate the binding energies. A twin anode (Mg and Al) X-ray source was operated at a constant power of 300 W using Al K radiation. The samples were sputter-cleaned in situ using a broad 3 keVAr + beam for 10 minutes. Due to the large dispersion of reference peak values found in the literature, XPS analysis of TiN, TiC and TiO 2 reference samples was performed to obtain experimentally the binding energies of interest. In Figure 1 the Ti2p spectra of the reference samples is presented clearly showing the doublets for TiC (454.6, 460.4 eV), TiN (455.4, 461.2 eV) and TiO 2 (458.5, 464.2 eV). In the TiN and TiC samples we also have found a doublet at (456.6, 462.3 eV) that some authors have associated to Ti 2 O 3 [18]. We have labeled it here as TiO x as it might include other titanium oxidation states. It is worth noting that the separation of the Ti2p3/2 and Ti2p1/2 components for the oxide doublets is 5.7 eV, while for the nitride and carbide is 5.8 eV. This is in very good agreement with the theoretical values expected. In Table 2 the binding energies of the C1s, N1s, Ti2p and O1s bands found for the reference samples are summarized and they will be used in the discussion of the XPS results.

Results and Discussion

Deposition Rate and Chemical Composition

The synthesis conditions together with the chemical composition and the thickness of the coatings are summarized in Table 1. The film thicknesses ranged from 1.4 to 3.2 µm. The deposition rate tends to increase with the increment of J Ti +Ag + J Ti , as it is clearly seen on Figure 2. However, for the first series a poisoning effect is observed in the sample with the highest current density. This is probably due to the simultaneous increase in the flow of reactive gases with the current density. In the inset on Figure 2, it is possible to observe the voltage increase in both targets during the deposition of the sample processed with J Ti +Ag + J Ti of 16 mA/cm 2 . The target voltage increase is due to the change of the target surface condition. In fact, due to the presence of reactive gases (N 2 + C 2 H 2 ), both TiN and TiC layers should be formed at the Ti surface targets (phenomenon called poisoning effect). Since the sputtering yield of both compounds is lower than that of Ti, a decrease in the deposition rate is consequently observed [19,20].

Concerning the others depositions no significant changes on the targets voltage values were observed during the deposition time. Also, it is possible to observe that the increment on the target voltage is higher for the TiAg target than for the Ti target. This effect cannot be explained from the formation of a silver nitride phase on the target surface, since silver is a poor nitride former. Depla et al. justify the increment on the target voltage during sputtering of silver target in an Ar/N 2 plasma, by the presence on non-reacted N atoms in the target top surface layers [21].

The chemical composition of the samples deposited onto silicon, measured by EPMA is also given in Table 1. The compositional analysis shows that by increasing the J TiAg /J Ti ratio for the first series the Ti content in the coatings decreased from 37 to 32 at. % along with an increase of the Ag content from 0 to 6 at. %. The C and N content do not change significantly with the synthesis conditions. In the 2 nd series, the increase of Ag area, promotes an increase in the silver contents in the coatings. The silver content changes from 11 to 27 at. % while Ti content decreased from 27 to 18 at. %. It can be seen that the current density applied to each magnetron (J TiAg and J Ti ) is the driven parameter for the film chemical composition. To that respect, Figure 3 shows the variation of the Ag/Ti ratio versus J TiAg /J Ti ratio. It is observed that in the second series, the raise of Ag/Ti ratio by increasing the current applied on Ti+Ag target is faster than in the first series. In the first series, this increase is lower due the smaller erosion area of Ag on Ti target. This means that it is not only the silver content that should control the films properties but also the Ag/Ti atomic ratio.

Phase Formation

XRD analysis was carried out in order to understand the evolution of the structure with the variation of Ag/Ti atomic ratio. The XRD patterns are shown in Figure 4 and the main identified crystalline phases are depicted, namely TiC 0.3 N 0.7 (ICDD card nr. 00-042-1488) and Ag (ICDD card nr. 00-004-0783). The differences in the chemical composition correlate well with the differences observed in the developed structure. In particular for the sample without silver the films crystallize in a B1-NaCl crystal structure typical for TiC 0.3 N 0.7 although a peak shift towards lower angles is denoted. This fact is in agreement with the measured chemical composition because these samples contain a slightly higher C/N atomic ratio than TiC 0.3 N 0.7 . Similar behavior was observed for the samples with low Ag/Ti atomic ratio (0.06 and 0.20), although a new diffraction peak attributed to Ag (111) begins to appear.

The samples with Ag/Ti atomic ratio 0.40 exhibit also a polycrystalline structure, clearly with formation of nanosized Ag clusters. For the Ag/Ti = 0.40 sample the structure is characterized by broader peaks in the range 35 to 45º. It is difficult to unequivocally identify which phases are formed. However, in agreement for the samples with low Ag/Ti atomic ratio there are fcc phases that have the main peaks close to these values (TiCN, and Ag). It is possible to infer that the XRD patterns reveal, at least, the development of a mixture of fcc-TiC 0.3 N 0.7 (clearly visible for lowest Ag/Ti atomic ratio) and fcc-Ag crystalline phases (clearly visible for the highest Ag/Ti atomic ratio). In fact, by increasing the Ag/Ti atomic ratio, the fcc-Ag phase becomes predominant for the Ag/Ti atomic ratio of 0.77. The increment of the Ag/Ti ratio leads to a loss of either the XRD peaks intensity and crystal size of the TiCN phase. These changes can be understood due to the interruption of the growth of TiCN phase by the nucleation of Ag nanocrystals and/or by the presence of an amorphous layer [22]. In fact, the low Ti/(C+N) ratio suggests the presence of an additional XRD amorphous phase, most likely of C or CN x type. Indeed, these phases were also observed within the TiCN, and ZrCN systems [1,3,23]. The presence of this amorphous layer was confirmed by Raman Spectroscopy and XPS analysis.

The XRD patterns of the Ag-containing films show, even for the lowest Ag contents, evidence of crystalline Ag presence. A small bump close to 38.12º (standard position of the (111) peak of fcc Ag phase) can be detected on the 0.06 Ag/Ti film, which becomes more and more evident with the Ag content increase until give rise to well defined and progressively narrower peaks. The grain size of TiCN and Ag phases was determined by Scherrer formula taking, in both cases, the (111) peak. The calculated values varied from 15 down to 9 nm and from 5 up to 8 nm, for TiCN and Ag, respectively, with the increase of Ag/Ti. This variation should be due to the simultaneous growth of Ag, TiCN and amorphous phases. During the deposition of TiCN phase the addition of Ag and/or the presence of the C-amorphous layer cause an interrupted nucleation of the crystals, leading to smaller crystal sizes [22,24]. However, due to the great mobility of Ag atoms, the increase of the Ag content progressively leads to higher Ag crystals, explaining the inverse trends of grain size for TiCN and Ag phases. In conclusion, the coatings should be imagined as a nanocomposite structure consisting of nanocrystals of both Ag and TiCN phases embedded in a C-rich amorphous matrix (incorporating N).

The nanocrystallinity of the Ag and TiCN grains can be confirmed by TEM crosssection observation. Fig. 5 shows the TEM micrograph of the coating with Ag/Ti = 0.40. A closer look at the Fig. 5 reveals nanocrystallites with sizes ranging from 5 to 17 nm. In the inset of this figure, a selected diffraction (SAD) pattern is presented. SAD pattern shows also diffuse rings where discrete spots are superimposed, revealing, as it would be expected, a nanocrystalline structure. Two different phases, both with a fcc structure were indexed. The first phase presents a lattice parameter (a = 0.429nm) very close to the TiC 0.3 N 0.7 phase (which lattice parameter strees free is 0.426 nm) while the second pattern can be indexed to Ag phase since the lattice parameter obtained by the SAD (a = 0.408 nm) it is close to this phase (a= 0.409 nm stress free). Meanwhile, the analysis of the Debye-Scherrer rings indicates growth on (111), ( 200), ( 220), ( 311) and (400) planes that can be assigned with the TiCN phase and also growth on (111), ( 200) and ( 220) planes for Ag phase.

SEM Analysis

The morphology of the coatings is disclosed by cross-sectional SEM micrographs of fractured samples and it is shown in fig. 6, for representative samples -Ag/Ti ratios of 0 (Fig. 6a 6a) shows a columnar-type structure with high porosity. The low temperature during the film growth (573 K), associated with low bombardment conditions, promotes the development of this kind of structure, due to the low adatom mobility. With the increase of a second crystalline phase (Ag) and the simultaneous growth of C(N) based amorphous phases, the films became slightly less porous. For the sample with Ag/Ti of 0.40 (Fig. 6c) it is possible to see the presence of Ag nanoclusters in the boundaries of the columns near the coatings surface, which was in some way predictable, once second phases usually tend to form in grain boundaries [22]. In the sample with Ag/Ti of 1.49 (Fig. 6d) is possible to see silver clusters and some aggregates in surface (inset on Fig. 6d).

XPS and Raman Spectroscopy

Raman Spectroscopy and XPS studies were performed on the Ag -TiCN samples, deposited on silicon substrates, with different Ag/Ti atomic ratios. XPS analysis was carried out with the aim of obtaining further information on the chemical bonding.

Figure 7 shows the a) Ti2p, b) C1s, c) N1s and d) Ag3d core level XPS spectra of the deposited Ag-TiCN films with Ag/Ti atomic ratio of 0, 0.20, 0.40 and 1.49.

In Figure 7a, there are three doublets clearly identified in the Ti2p spectra of all samples. The first two doublets with Ti2p3/2 components centered at 458.5 and 456.6 eV respectively can be straightforwardly ascribed to TiO 2 and TiO x bonds for all samples as derived from the analysis of the reference samples (see Figure 1 and Table 2). The oxygen impurities exeunt due to residual oxygen in the vacuum chamber, in the targets and also due to contamination after deposition. For sample without silver the third contribution is found to be at 455.1 eV (for the Ti2p3/2 component), that is in between the TiC (454.6 eV) and TiN (455.4 eV) bonds. This suggests the formation of a TiCN phase in good agreement with the XRD results previously shown. Similar behavior is found for sample with Ag/Ti = 0.20. However, as the silver ratio increases there is a slight but progressive shift of this third doublet towards higher binding energies. In sample with Ag/Ti = 0.40 the third doublet is centered at 455.2 eV while for Ag/Ti = 1.49 it is at 455.4 eV, namely the binding energy of TiN. This shift is therefore ascribed to a continuous decrease of the titanium bonded to carbon. The Gibbs free energy for the formation of TiN phases is -290 kJ/mol and for TiC is -180 kJ/mol [25]. Therefore, with the decreasing of Ti content, there is a clear trend to the formation of the thermodynamically more stable nitride phases. In fact when analyzing the C1s spectra in Figure 7b, the intensity of component ascribed to the C-Ti bonds at 282 eV decreases with the increase of the Ag/Ti ratio. In particular for sample with Ag/Ti = 1.49 this contribution simply disappears. On the other hand, for samples with high Ag/Ti ratio there is an increase of the intensity of the component ascribed to C-N bonds (286.9 eV), which is parallel to the behavior of the component assigned to N-C bonds in the N1s spectra (see below), reinforcing the idea of the formation of an amorphous Cphase incorporating N. In the N1s spectra of Figure 7c, two contributions were found at 397.0 eV and 399.2 eV ascribed to N-Ti and N-C bonds, respectively (see Table 1). The intensity of the N-C component becomes significant when the Ag/Ti increases (in agreement with the results for C1s spectra). Therefore, we can state that the presence of CN bonds is observed in the samples with Ag/Ti atomic ratios of 0.40 and 1.49 and the appearance of these bonds are attributed to the decrease of Ti content in the coatings.

The excess of C and N (low Ti/C+N) ratio, as it is possible to confirm by the composition results) promotes the formation of CN amorphous phases detected in both XPS and Raman Spectroscopy analysis (see below). Finally, from the analysis of Ag 3d photoelectron spectra (Figure 7d) it was possible to confirm that the only contribution is the Ag-Ag metal bonds, which was somehow predictable due to the low solubility of C and N in silver.

Figure 8 shows the Raman spectra of deposited Ag-TiCN samples with Ag/Ti atomic ratios 0, 0.20, 0.40 and 1.49 . The aim of Raman Spectroscopy analysis was to confirm the interpretation of structural and chemical bonding aspects done by XPS and XRD. As already discussed from the XRD analysis it is possible to observe the presence of broad peak between 35 and 45 º for the sample with Ag/Ti atomic ratio of 0.20 and 0.40, which may be associated to the contribution of different nanocrystalline phases: TiCN and Ag, which presents three diffraction peaks between 36 and 38,6º (ICDD card nr. . By the literature for TiN four peaks at approximately 225 (TA), 315 (LA), 450 (2A) and 550 cm -1 (TO), and for TiC four peaks located at 280, 385, 585 and 675 cm -1 are identified in the Raman spectra [1,3,26,27]. According to our results (Fig. 8) seems that the TiCN bonds should be located between TiN and TiC peaks.

In the region between 1250 and 1595 cm -1 several authors assigned carbon phases like C-N at 1250cm-1 and C=N at 1500 cm-1 as well as D and G bands of carbon materials at 1370 and 1580 cm-1 [23,28,29]. Due to the similarity of the vibrational frequencies of C-C and C-N the interpretation of the results is difficult, due to the appearance of broad bands. Nevertheless, the Raman band around 720 cm -1 , that starts to appear in the sample with Ag/Ti atomic ratio of 0.40, is also associated to the presence of C-N bonds [3], which allows confirming the presence of N in the amorphous CN phases as predicted from XPS results.

Conclusions

Ag-TiCN thin films were deposited by reactive magnetron sputtering with different density of currents applied to the Ti and Ti+Ag targets, which controls the chemical composition of the samples studied. The structure evolution is correlated with the Ag/Ti atomic ratio. Samples with low Ag/Ti atomic ratio (<0.20), crystallized in a B1-NaCl crystal structure typical of TiC 0.3 N 0.7 . With the increase of Ag/Ti atomic ratio a second crystalline phase begins to appear promoting a reduction in the grain size and a densification of the coatings. In samples with intermediate Ag/Ti ratio (0.20 and 0.40) the increase of silver content promotes a competitive growth between Ag and TiC 0.3 N 0.7 phases. Simultaneously, amorphous carbon based phases begin to appear. Silver phases and CN tends to segregate in the grain boundaries of TiC 0.3 N 0.7 causing a decrease in the grain size and a densification of the coatings. In samples with high Ag/Ti atomic ratio (>0.77), Ag appears as the dominant crystalline phase. According to the results obtained in XPS analysis, the TiCN bonds are replaced by TiN bonds as consequence of the decrease of Ti content with the increase of Ag/Ti atomic ratio. The presence of CN amorphous phases was confirmed by XPS and Raman Spectroscopy analysis.

  ); 0.20 (Fig 6b); 0.40 (Fig 6c) and 1.49 (Fig 6d). The cross-section of the sample without silver (Fig.

Fig 1 .Fig. 3 .

 13 Fig 1. XPS spectra of Ti 2p core level of TiN, TiC and TiO 2 reference samples

Fig. 5 .

 5 Fig. 5. TEM micrograph from the cross-section of the Ag-TiCN coatings with Ag/Ti = 0.40 Fig. 5 b) Diffraction pattern from a selected area.

Fig. 6 .

 6 Fig. 6. Cross-sectional SEM micrographs for different atomic ratios Ag/Ti: a) 0 b) 0.20 c) 0.40 d) 1.49 Inset on Fig 6d) SEM image from the sample surface where are visible Ag clusters.

Fig. 7 .Fig. 8 .

 78 Fig.7. XPS spectra of a) Ti 2p b) C 1s c) N 1s d) Ag 3d core levels of the Ag-TiCN coatings deposited by DC reactive magnetron sputtering with different Ag/Ti atomic ratios

Figure 5 (

 5 Figure 5 (Fig5corr.tif)

Acknowledgements

The authors are grateful to Dr. Isabel Muñoz Ochando (Instituto de Ciencia y Tecnología de Polímeros ICTP-CSIC) for her assistance in carrying out the Raman spectroscopic analysis. The work was financially supported by Portuguese national funds through the CRUP Institution (project "Acção Nº E -1007/08) and FCT-Fundação para a Ciência e a Tecnologia, (project PTDC/CTM/102853/2008) and by the Spanish Ministry of Science and Innovation (projects FUNCOAT CSD2008-00023 and MAT2008-06618-C02). REG wish also to thank the MCINN for the financial support within the Ramón y Cajal programme. This research was partially sponsored by FEDER funds through the program COMPETE-Programa Operacional Factores de Competitividade.


S R Frater

J P Huisman

J L Korterik

W L Herek

Vos

G Ctistis
email: g.ctistis@utwente.nl

E H Frater

S R Huisman

J P Korterik

J L Herek

L Vos

P W H Pinkse
email: p.w.h.pinkse@utwente.nl

Controlling the quality factor of a tuning-fork resonance between 9 K and 300 K for scanning-probe microscopy

Keywords: numbers: 07.79.Lh, 07.79.Fc, 68.37.Ps, 68.37.Uv

Controlling the quality factor of a tuning-fork resonance between 9 and 300K for scanning-probe microscopy

G Ctistis, E H

Introduction

Quartz crystal oscillators are widely used as a frequency reference. The mechanical resonance is intrinsically stable and the crystal's piezo-electric effect allows for direct electronic interfacing. Quartz tuning forks have proven to be extremely useful as sensors for temperature [1,2], mass [3], pressure [7,5,6,4], and friction [8,9,10,12,13,15,14,11,17,16]. Due to their high mechanical quality factor Q between 10 3 to 10 5 they are very sensitive to environmental changes. Sub-pN forces dragging on their prongs can easily be detected. The dragging forces change the oscillating properties of the tuning fork through a change in the spring constant. This gives rise to a shift in the resonant frequency and a corresponding change in the amplitude of the vibrating tuning fork. This change in the vibration amplitude changes the current through the tuning fork, which is generated through the piezoelectric effect. Thus, by measuring the changes of the electric signal, which can be measured very accurately, one can diagnose the prevailing forces.

This sensitivity has made quartz tuning forks become popular in two fields of research: temperature measurement of liquid He, where the resonance frequency is a measure for the temperature of the surrounding liquid [18,19,20] and in scanningprobe microscopy, where the forces between tip and sample are used to image surfaces with atomic resolution [8,9,10,21]. More and more low-temperature scanning-probe microscopes are used nowadays [22,23,24] where the height of the tip is controlled via the shear forces of a vibrating tip attached to a tuning fork. An accurate control of the resonance response of the tuning fork is therefore highly desired because the performance of the height control critically depends on the resonance properties of the tuning fork. These properties are thereby described by the quality factor Q of the resonance. It is defined as 2π times the mean stored energy divided by the work per cycle or equivalently by Q = ω 0 /∆ω, where ω 0 is the resonance frequency and ∆ω the width of the resonance. A high quality factor Q normally results in an undesireably slow response of the system [25] since the response time τ of the system is related to the quality factor by τ = 2Q/ω 0 . Controlling the quality factor of the resonance provides a solution to this problem. It has already been shown [25,26,27,28,29,30,31] that quality factor control is possible. Yet, up to now, it has only been applied to measurements in liquids, in ultrahigh vacuum, or at liquid He temperatures, but it has not been used to measure temperature-dependent system dynamics.

Here, we present the dynamic control of the quality factor of the resonance of a tuning fork over a large range of temperatures. We are able to adjust the quality factor of the resonance to a desired value and keep it constant while changing the temperature, which is vital for applications purpose in near-field scanning optical microscopy (NSOM).

The outline of the paper is as follows: a brief introduction into the oscillator tuningfork system and its temperature dependence is followed by a description of the quality factor controlled system and the presentation of the data showing that we are able to control the quality factor of the resonance over a wide range of temperatures. A short conclusion with prospects for possible experiments closes the paper.

The tuning fork system

The experiments were performed both under ambient conditions as well as in a cryostat at a temperature between 9 K and 300 K and a base pressure of less than 1 × 10 -4 mbar. Figure 1 shows a schematic of the electric circuit to measure the electric response of the tuning fork. A sinusoidal signal from an external function generator (IN) drives the tuning fork at a constant frequency with a constant amplitude. The resulting oscillation amplitude and phase shift are detected by a lock-in amplifier (OUT). The constant frequency of the function generator acts also as a reference for the lock-in. The additional feedback circuit (gray dashed box) consisting of an amplifier and a phase shifter (e.g. a fixed phase of 90 • ) is responsible for the quality-factor control and works as described for example in Ref [26]. The signal of this feedback circuit is amplified (gain) and used to excite the tuning fork in addition to the signal from the function generator (Σ). The phase shifter controls thereby the time delay (phase) between the excitation and the tuning-fork signal, which results in a damping or amplification of the tuning-fork excitation. If the driving signal and the control signal have a phase difference of π/2 we can cancel the natural damping of the tuning fork. By adjusting the gain factor g and the phase (corresponding time delay t 0 ) we can increase or decrease the quality factor Q eff [29,31]:

Q eff (g, t 0 ) = 1 1/Q -g sin (ω 0 t 0 ) . (1)
The oscillator system itself consists of a standard quartz tuning fork with an eigenfrequency of ω 0 ≈ 2π × 2 15 Hz. One prong is l r = 3.9 mm long, t r = 600 µm thick, and w r = 330 µm wide. Since the tuning fork is excited electrically, we expect the so-called anti-phase mode to be the dominant oscillation mode [32,33,34]. In part of the experiments an optical fiber was glued to one prong of the tuning fork with epoxy glue for the use in temperature-dependent NSOM measurements. This changes not only the thickness of one prong by approximately half of the cross-sectional area of the fiber (t = 617 µm) but also changes the mass of this prong compared to the second unloaded prong. The tuning fork is therefore not balanced anymore but asymmetric. One expects changes in the response of the tuning fork as compared to a bare one as has already been reported before [32,33,34]. The elastic constant of quartz and its density are E = 8.68 × 10 10 N/m 2 and ρ = 2649 kg/m 3 , respectively. [START_REF]CRC Handbook of Chemistry and Physics[END_REF] The resonance frequency of the tuning fork-fiber system is calculated using: [START_REF]Scanning force Microscopy 1991[END_REF]

ν 0 = E 4 t r ( wr lr ) 3 αρl r t r w r = 33690 Hz (2)
The measured eigenfrequency of the tuning fork-fiber system is ν meas = 33550 Hz. The calculation matches the experimentally determined frequency very well given the slight difference in values due to the evaporated contacts, the glue, and the attached fiber.

Figure 2 shows the amplitude (upper panel) and the phase (lower panel) response of a bare tuning fork (black circles) as well as of a tuning fork-fiber system (green squares). Both resonance curves were taken under ambient conditions. It is clearly visible that gluing a fiber to one prong of the tuning fork changes the oscillator drastically. The resonance blue-shifts by 1 kHz and its amplitude is damped. The dashed lines in Fig. 2 show the resonances' amplitude and phase response, which is calculated with:

A(ω) = A 0 m 1 (ω 2 0 -ω 2 ) 2 + γ 2 ω 2 (3)
φ(ω) = arctan ( γω ω 2 0 -ω 2 ), (4)
where ω 0 is the resonance frequency, m is the mass of the system, A 0 the driving amplitude, and γ the damping. The model matches the experiment well and reveals quality factors Q = ω 0 /∆ω of 5400 for the bare tuning fork and 500 for the tuning fork-fiber system, respectively.

In the next step we determined the temperature dependence of the resonance response of the bare tuning fork and the tuning-fork-fiber system. We transferred the oscillator system into a small cryophysics cryostat with a model 22CTI cryodyne closed-cycle helium cooler, which allowed us to measure at temperatures between 9 and 300 K. Figure 3 shows the acquired data in a color-scale representation where the colors represent the amplitude of the resonance measured versus the frequency for each temperature. The temperature-dependent response of the bare tuning fork (lower curve) shows two major features. First of all only a small shift of the resonance frequency is measured throughout the whole temperature regime. Down to a temperature of about 70 K this frequency shift is well described by:

ω = ω 0 [1 -α • (T -T 0 ) 2 ], (5)
with α = 3 × 10 -8 K -2 and T 0 = 300 K. The value for α corresponds thereby with the value given by the manufacturer [37] and is inherent to the quartz crystal and to the orientation in which it is cut. Below 77 K the resonance shift is flattened, which is mainly due to the change in the elastic constants of the quartz [START_REF] Fukuhara | [END_REF]. Second, the resonance stays very sharp throughout the whole temperature range and has a quality factor of Q ≈ 14000 (see also Fig. 4). We can assume that the measured temperaturedependent response of the bare tuning fork is universal due to the manufacturer's design. The effect of the vacuum in the cryostat on the resonance response can be neglected in our experiments because it leads to a relative shift of the resonance in the order of 10 -4 . In contrast to the bare tuning fork, the temperature dependence of the resonance response of the combined tuning fork-fiber system is more complex (Fig. 3, upper curve). This is caused by the glue and the attached fiber, which lead to a lower quality factor compared to the bare tuning fork. Overall, a blue-shift of the resonance frequency can be observed, which corresponds well with an increased stiffness of the system. More evident is that the width of the resonance decreases with decreasing temperature. This means that at low temperatures the oscillator system has a higher quality factor than at room temperature. Another feature that is apparent in the resonance response is at T ≈ 60 K. One can clearly observe a splitting and anti-crossing behavior of the resonance where the two branches interchange oscillator strength, i.e. amplitude. Whereas the dominant mode is the fundamental anti-symmetric mode of the tuning fork, the second mode cannot be the fundamental symmetric mode because the symmetric and anti-symmetric modes are at least 10 kHz apart [33]. Even considering the asymmetry of the tuning fork because of the loaded prong using a two-coupled-oscillator model [32,33,34] cannot fully explain the observed resonance splitting. An explanation might be that the oscillation mode of the loaded prong alone changes with temperature due to the changing elastic properties of the glue that was used to attach the fiber. This assumption is strengthened by the fact that the observed splitting strongly depends on the amount of glue used, the mass of the fiber attached, and how the fiber is attached to one prong of the tuning fork, thus showing a case-by-case response. A detailed analysis of the response is beyond the scope of the present paper.

Controlling the quality factor

It is evident from these measurements that in order to perform shear-force controlled scanning-probe measurements, we need to control the quality factor of the resonance and keep it stable in the complete temperature range. As a rule of thumb the quality factor of a tuning-fork system used in scanning probe microscopy (SPM) should in general lay between 300 and 1000. In this range the feedback system for the shearforce measurements is found to be well balanced, allowing for stable measurements.

From our data shown in Fig. 3 we can extract the corresponding quality factors for our systems from the resonance width and the resonance frequency of the tuning-fork response. Throughout the whole temperature range these values are shown in Fig. 4 as open symbols, squares for the bare tuning fork and circles for the tuning-fork-fiber system. It is seen that the bare tuning fork has quality factors exceeding Q = 12000. More striking is the dependence for the tuning-fork-fiber system. It is obvious that in the high-temperature region the quality factor is in the desired range (Q ≈ 500). Yet, by decreasing temperature the quality factor increases by a factor of ∼ 5 to Q = 2300 at 9 K. Thereby the change in quality factor is not monotonous but changes also due to the observed mode anti-crossing. The filled symbols in Fig. 4 display our results after applying our electronic phase compensation, i.e., our quality factor control. We succeeded in reducing the quality factor of both the bare tuning fork and the tuning-fork-fiber system and kept it nearly constant throughout the whole temperature range.

To test our system's dynamic performance we continuously changed the gain settings for the quality-factor control. Figure 5 shows the resonance response of a tuning fork-fiber system at a temperature of 9 K. We recorded the amplitude (upper panel) as well as phase (lower panel) versus the frequency. The starting value of Q = 2300 (compare also Fig. 4) is achieved without any quality factor control. As is evident from the graph, we can change the quality factor of the tuning fork smoothly down to Q = 40 by changing the gain in the phase shifter of the quality-factor-control unit (Fig. 1, gray box). The good performance of our quality factor control is also apparent in the recorded phase, a smooth transition from high-to low-Q is visible.

The only restricting factor still prominent in the measurements is the noise level. For lower quality factors the signal-to-noise level is the limiting parameter to resolve the resonance of the tuning fork clearly. If the main noise source is the electronics that were used, this restriction can be reduced significantly. Applying an electronic compensation scheme where the reference signal and the signal from the tuning fork are subtracted, we achieved to reduce the noise level by at least a factor of 10 (data not shown here), thereby increasing the performance of the system for the entire range of quality factors. x-Position (µm) With the uncontrolled high value for Q we could not achieve any shear-force controlled measurement of a topography. Yet, by changing the quality factor and keep it constant (Q ≈ 350) we are now able to measure the topography of a standard test grating [START_REF] Co | TGQ1 test grating[END_REF] with our tuning fork-fiber system, as shown in Fig. 6 (left panel). The displayed squares have a height of 20 nm, a pitch size of 1.5 µm, and a periodicity of 3 µm. There is a difference between the left and right part of the recorded image visible. The left part has more noise along the fast scan direction of the microscope. Changing the gain of the height feedback halfway during the scan led to reduced noise and a higher contrast in the topography. This is visible in more detail in the cross sectional scan (Fig. 6, right panel) along the dashed line of the topography image. The height of the pitches deduced from this scan matches the height given by the manufacturer and shows the good performance of the shear-force quality-factor controlled scanning system.

Conclusions and Outlook

We presented our results on the dynamic control of the quality factor of a quartz tuning fork in a large temperature range. We showed that we are able to compensate for the temperature-dependent change in the resonance response of a tuning fork. Moreover, we applied our compensation to a tuning fork-fiber system and achieved to keep the quality factor in a regime where shear-force measurements are possible throughout the whole temperature range. We are also able to smoothly tune the quality factor at each temperature to a certain value. To be able to measure topography with the same quality factor of the tuning fork is a major step to a more efficient and straightforward use of such scanning-probe microscopy systems and allows their use over the complete temperature range as well as in all sorts of environments.

As a result of our experiments, dynamic temperature-dependent near-field optical measurements have become possible. The method can be used, e.g., to measure locally resolved structural phase transitions, such as domain formation, by optical means with varying temperature and thus deduce the optical properties of sub-wavelength structures near a transition point. Furthermore, the temperature dependence of light propagation in nanophotonic objects can be studied.

Figure 1 .

 1 Figure 1. Schematic of the electric circuit for the tuning-fork excitation. The parallel amplifier and capacitance are for compensation of the parasitic capacitance of the tuning fork. The dashed box shows the part responsible for the quality-factor control, consisting of a 90 • phase shifter (Q-control) and an amplifier (gain).

Figure 2 .

 2 Figure 2. (color online) Amplitude (upper panel) and phase (lower panel) response of a bare tuning fork (circles) and a tuning fork with a glued fiber (squares) at room temperature. Additionally, fitted curves (dashed lines) determining the quality factors of the two tuning forks are shown. The black dotted lines indicate the center frequency.

Figure 3 .

 3 Figure 3. (color online) Measurement of the amplitude of the oscillation of a bare tuning fork (lower curve) and of a tuning fork with an attached fiber (upper curve) versus frequency and temperature. The change of the resonance frequency and width are significant in the second case.

Figure 4 .

 4 Figure 4. (color online) Measured quality factor versus temperature for a bare tuning fork (open squares) and a tuning fork with a glued fiber (open circles). The shaded region indicates the desired range of quality factors where shear-force measurements are possible. Applying quality-factor control results in a near-constant quality factor for both systems (filled symbols).

Figure 5 .

 5 Figure 5. (color online) Quality-factor control measurements of the resonance at T = 9 K for a tuning fork with an attached fiber. (Upper panel) Amplitude response of the tuning fork. The quality factor of the resonance was tuned between Q = 40 and 2300. (Lower panel) Simultaneously recorded phase of the oscillator.

3 9 Figure 6 .

 396 Figure 6. (color online) Shear-force topography scan of a test grating at room temperature. For this scan an adjusted quality factor of Q ≈ 350 was used. Between the left and right parts of the image the gain in the height feedback control was changed. (right panel) The cross section was taken along the dashed line in the topography image.

Acknowledgments

We would like to thank Frans Segerink, Allard Mosk, Herman Offerhaus, Cock Harteveld, and Dick Veldhuis for discussions and help with setting up equipment. PP acknowledges financial support by FOM Projectruimte. GC acknowledges financial support by NWO-Nano. This work is also part of the research program of FOM, which is financially supported by NWO.


Maria Paola Lombardi

Raoul Cm Hennekam
email: r.c.hennekam@amc.uva.nl

Saskia Bulk

Jacopo Celli

Anne Lampe

Michael T Gabbett

Lillian Bomme-Ousager

Jasper J Van Der Smagt

Maria Soller

Eva-Lena Stattin

Lillian Bomme Ousager

Eva- Lena Stattin

Marcel A M M Mannens

Robert Smigiel

Raoul C Hennekam

Dr Rcm Hennekam

Mutation Update for the PORCN Gene

Keywords: Goltz Gorlin syndrome, PORCN, FDH, LOVD database

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Goltz-Gorlin Syndrome (GGS) or Focal dermal hypoplasia (MIM 305600) is an X-linked dominant disorder characterized by a combination of malformations and dysplasias, mainly affecting the skin (linear hypoplastic areas; linear hyperpigmented and hypopigmented areas, fat herniation through skin; papillomas around body orifices), skeleton (oligodactyly; ectrodactyly; syndactyly; polydactyly; caudal appendage; extreme variability) and eyes (microphthalmia; coloboma; cataract; corneal opacities) [START_REF] Gorlin | Focal dermal hypoplasia syndrome[END_REF][START_REF] Gorlin | Focal dermal hypoplasia syndrome[END_REF]. A wide variety of other manifestations has been reported. Goltz-Gorlin syndrome is an infrequently reported disorder: about 250 cases are reported in literature [START_REF] Hennekam | Gorlin's Syndromes of the Head and Neck[END_REF].

Estimates of the prevalence are not available.

In 2007 two groups independently described for the first time mutations in PORCN as being responsible for GGS [START_REF] Grzeschik | Deficiency of PORCN, a regulator of Wnt signaling, is associated with focal dermal hypoplasia[END_REF][START_REF] Wang | Mutations in the X-linked PORCN, a putative regulator of WNT signaling, cause focal dermal hypoplasia[END_REF].

Subsequently it was found that PORCN mutations can also be found in the pentalogy of Cantrell and the limb-body wall complex [START_REF] Maas | Phenotype and genotype in 17 patients with Goltz-Gorlin Syndrome[END_REF] and deletions can cause angioma serpiginosum [START_REF] Houge | An Xp11.23 deletion containing PORCN may also cause angioma serpiginosum, a cosmetic skin disease associated with extreme skewing of X-inactivation[END_REF]. PORCN (isoform D NM_203475.1), located at Xp11.23, encodes a 52 kDa protein of 461 amino acids.

PORC proteins are predicted to be putative O-acyltransferases involved in palmitoylation of Wnt signaling proteins, which have key roles in embryonic tissue development [Paller, 2009;[START_REF] Clements | Importance of PORCN and Wnt signalling pathways in embryogenesis[END_REF]. Similar to many other proteins in the PORC family PORCN has multiple transmembrane domains, predicted to traverse the membrane of the endoplasmic reticulum, and one large membrane bound Oacyltransferase domain (aa 115-402) [Hoffmann K, 2000; http:www.uniprot.org/uniprot/Q9H237]. Here, we present a review of all 73 PORCN mutations published so far, and report 7 new mutations, including 6 intragenic mutations and one microdeletion. In addition we announce a web-based locus specific database that has been created to list all identified variants and which allows the inclusion of future reports. The database is based on the Leiden Open (source) Variation Database (LOVD) software [START_REF] Fokkema | LOVD: easy creation of a locus-specific database using an "LSBD-in-a-box" approach[END_REF]. In the PORCN-LOVD database emphasis is given to clinical data of each individual patient. Comparisons within the database should enable colleagues world-wide to study genotype-phenotype correlations in detail.

PORCN database construction

We performed a PubMed search using as search terms PORCN, Goltz-Gorlin syndrome, Focal Dermal Hypoplasia, Angioma Serpiginosum and pentalogy of Cantrell. The reference lists of all papers retrieved this way were hand searched for additional publications. Furthermore, we have been offering PORCN mutation analysis on a diagnostic basis from 2007 on and added all PORCN mutations detected ourselves since then. Part of our results has been published elsewhere [START_REF] Maas | Phenotype and genotype in 17 patients with Goltz-Gorlin Syndrome[END_REF].

All information gathered on mutations and mutation carriers were initially stored in individual fields in an offline Microsoft Excel worksheet (Microsoft, Redmond, WA) and subsequently converted into a Web accessible PORCN gene variant database within the Leiden Open Variation Database (LOVD) platform [START_REF] Fokkema | LOVD: easy creation of a locus-specific database using an "LSBD-in-a-box" approach[END_REF]. This database has also been included in the Human Genome Variation Society (HGVS) list of LSDBs. In the database information are section the presence or absence of all major clinical manifestations of Goltz-Gorlin syndrome is reported in addition to gender, geographic / ethnic origin, times reported and patient remarks. Additional information on the pathogenicity of each variant, in the format 'reported/concluded', are given in a separate field, which can be visualized by using the 'View all contents' feature of the database.

The database can be consulted at www.lovd.nl/PORCN and will be updated with data from literature as well as with the unpublished variants identified in our diagnostic unit. Furthermore, all scientists/clinicians are invited to submit their unpublished data to the database using an online submission form. Submissions will be checked by the curators (M.P.L.; R.C.H.) and completed if necessary. The unpublished status will be clearly indicated, as well as the identity of the submitter.

Mutations in the PORCN gene

To date (September 2010), 106 GGS patients with an identified PORCN mutation are registered in the PORCN-LOVD database. Ten are males having point mutations identified in a mosaic status. All mosaic mutations were detected in fibroblasts and not in peripheral blood leucocytes, except in one patient (database ID_PORCN_00080; [START_REF] Vreeburg | Focal dermal hypoplasia in a male patient due to mosaicism for a novel PORCN single nucleotide deletion[END_REF]. In total 80 unique mutations have been reported. Mutations are named according to HGVS nomenclature guidelines reported mutations have been renamed accordingly but the original nomenclature is also included in the corresponding entry to allow cross-reference. All mutations listed in the database are predicted to be pathogenic either based on the nature of the mutation or on the location in a functional domain.

Sixty-eight unique point mutations or small insertions/deletions have been detected so far (Table 1). Six mutations, p.Met1?, p.His252Tyr, p.Trp305X and the splicing mutations c.845+1G>C, c.947-1G>C and c.1024-2A>G , are reported here for the first time. The major clinical findings associated with these mutations, are provided in Table 2 as presented in the LOVD database in the 'Patient data' section, and compared to the data of the other patients in the database.

In Table 2 the available major clinical findings of all patients with an identified PORCN mutations are summarized, based on the mutation type.

The distribution of all mutations in PORCN is indicated in Figure 1. Mutations are scattered along the coding sequence, with the exception of exons 6 and 7, where no mutation have been found to date. Also the type of mutations is variable: nonsense mutations are the most represented (n=20), followed by missense (n=18) mutations. In addition there were 16 frameshift, 10 splicing and 4 in frame insertions/deletions. The majority of all PORCN mutations (46/68) are predicted to lead to protein truncation and loss of function. There are 11 recurrent mutations which are mostly transitions at CpG sites. Three mutations, p.Arg124X, p.Arg365Gln and p.Arg243X are particularly common and occurred in 5 (p.Arg124X, p.Arg365Gln) and 4 (p.Arg243X) patients, respectively. These mutations occurred also in patients with various ethnic backgrounds, suggesting that they may represent mutational In general however, the information available on the ethnic background and/or country of origin of patients is limited and analysis of geographical distribution of mutations or of their incidence in the various ethnic groups is not possible at present.

In 12 patients large deletions have been identified by array-CGH or MAPH analysis: 11 patients have deletions of the whole gene, in most cases encompassing the neighbouring genes, and in a single patient a partial gene deletion encompassing exon 1 to exon 4 was present (Table 3, Figure 2). As the exact location of the breakpoints of some deletions has not been determined, it remains uncertain whether some of these represent recurrent events. X-inactivation data were available for all patients except one and showed extreme skewing in all available patients.

Clinical and Diagnostic Relevance

The identification of mutations in PORCN confirms the clinical diagnosis in patients suspected of having GGS. In addition a molecular diagnosis allows for reliable genetic counselling and prenatal diagnosis if desired. Germ-line mosaicism for PORCN mutations has been described [START_REF] Maas | Phenotype and genotype in 17 patients with Goltz-Gorlin Syndrome[END_REF]. Our lab has been offering molecular genetic testing for PORCN mutations since 2007 with a combined approach based on direct sequencing and MLPA analysis. We analysed 40 index patients and found mutations in 25 patients (62.5%), including one deletion of the entire PORCN gene. The lack of mutations in some patients is in part explainable as several samples were submitted 'to exclude GGS'. In all patients evaluated by the present senior author as being classically affected a mutation has been found [START_REF] Maas | Phenotype and genotype in 17 patients with Goltz-Gorlin Syndrome[END_REF]. Still, the existence of phenocopies can not be excluded. Thus far no defects in other genes have been potentially correlated to the GGS phenotype, [START_REF] Houge | An Xp11.23 deletion containing PORCN may also cause angioma serpiginosum, a cosmetic skin disease associated with extreme skewing of X-inactivation[END_REF], limb-body wall complex, and pentalogy of Cantrell [START_REF] Maas | Phenotype and genotype in 17 patients with Goltz-Gorlin Syndrome[END_REF] (Table 1).

The phenotypic variability, the small number of patients known so far which are fully clinically characterised, and the lack of information on their clinical presentation might have caused misdiagnoses.

One of the major goals of the establishment of the PORCN_database is to collect detailed information on the clinical findings in Goltz-Gorlin patients and to harmonize the clinical criteria for diagnosis. At present, a description of the positively present clinical features is available for most but not all patients. However we scored features as present only if stated in the text of published cases or clearly visible in figures. Therefore there will be a bias towards the number of positive manifestations in patients in the database hampering reliable genotype-phenotype correlations.

Registration of clinical findings according to common criteria, as required in the database, will help harmonizing patients' phenotypic description.

One characteristic that is consistently found in all patients with a PORCN mutation is the presence of linear skin lesions / skin hypoplasia reported in 91 out of 92 patients with documented clinical findings. Other features that appear to be prevalent upon documentation are skeletal defects (a limb anomaly was present in 83 patients) and the thin and protruding ears.

Data on X-inactivation were available for all patients except one with large rearrangements. Large deletions were associated in all available patients with an extremely skewed X-inactivation pattern. Three of the 11 patients of whom data were available showed a mild phenotype. This confirms the observation made by Houge et [START_REF] Houge | An Xp11.23 deletion containing PORCN may also cause angioma serpiginosum, a cosmetic skin disease associated with extreme skewing of X-inactivation[END_REF].

Otherwise genotype-phenotype correlations have been elusive so far.

Classically affected Goltz-Gorlin patients have been reported with additional defects (midline thoracic and abdominal wall) [START_REF] Maas | Phenotype and genotype in 17 patients with Goltz-Gorlin Syndrome[END_REF] which are also found in the pentalogy of Cantrell and the limb-body wall complex [Van Allen et al., 1987;[START_REF] Cantrell | A syndrome of congenital defects involving the abdominal wall, sternum, diaphragm, pericardium and heart[END_REF][START_REF] Smigiel | Co-occurrence of Severe Goltz-Gorlin Syndrome and pentalogy of Cantrell_Case report and review of the literature[END_REF] Xp11.23 is drawn as in www.ensembl.org/Homo_sapiens/Location/Overview?db=core;g=ENSG0000010231 2;r=X:48367350648379202

F o r P e e r R e v i e w

  Data and Patient Data. The 'variant data' section contains the following variant-specific fields : exon / intron location, DNA / RNA/protein change, X chromosome inactivation pattern, database ID, variant origin, variant remarks, reference, template / technique used for detection and frequency. In the 'patient data'

  HGVS.org) and numbered with respect to the PORCN gene reference sequence (NG_009278.1 covering PORCN transcript NM_203475.1). Previously

  PORCN have been found associated with additional phenotypes: angioma serpiginosum

  the variability of the phenotype depending on the efficacy of the protective effect exerted by X-inactivation

Figure 1 .Figure 2 .

 12 Figure 1. Gene map of distribution of point mutations in the PORCN gene. The boxes

  Figure 1 254x190mm (96 x 96 DPI)

Table 1 . Variants listed on the PORCN Database

 1

	Database_ID	Gender	Exon	Mutation	Protein Change	Type	References	Disease
	PORCN_00001	F	02	c.1A>G	p.Met1? ¶	change of	this report	#	GGS
	PORCN_00002	F	02	c.3G>A	p.Met1Ile* ± ¶	translation initiation codon Bornholdt et al, 2009 GGS
	PORCN_00003	F	02	c.49dupT	p.Cys17LeufsX11	frameshift	Harmsen et al, 2009	GGS
	PORCN_00004	F	02	c.53dupT	p.Leu18LysfsX10*	frameshift	Wang et al, 2007	GGS
	PORCN_00005	F	02	c.74delG	p.Gly25AlafsX27	frameshift	Clements et al, 2009 GGS
	PORCN_00006	F	03	c.166_167dupAC	p.Val57ProfsX60	frameshift	Harmsen et al, 2009	GGS
	PORCN_00007	F	03	c.178G>A	p.Gly60Arg	missense	Wang et al, 2007	GGS
	PORCN_00008	F	03	c.221G>A	p.Trp74X	nonsense	Harmsen et al, 2009	GGS
	PORCN_00009	F	03	c.222G>A	p.Trp74X	nonsense	Grzeschik et al, 2007 GGS
	PORCN_00010	F	03	c.268C>T	p.Arg90X	nonsense	Wang et al, 2007
		F					Bornholdt et al, 2009 GGS
		F					Harmsen et al, 2009
	PORCN_00011	F	03	c.283C>T	p.Arg95X	nonsense	Bornholdt et al, 2009 GGS
		F					Maas et al, 2009
	PORCN_00012	M	04	c.370C>T	p.Arg124X	nonsense	Wang et al, 2007
		F					Bornholdt et al, 2009 GGS
		F F					Grzeschik et al, 2007 this report §
	PORCN_00013	F	04i	c.373+1G>A	-	splicing	Leoyklang et al, 2009 GGS
	PORCN_00014	F	04i	c.374-1G>A	-	splicing	Bornholdt et al, 2009 GGS
	PORCN_00015	F	04i	c.374-15T>A	-	splicing	Schaffer et al, 2009	GGS
	PORCN_00016	F	04i	c.374-46T>A	-	splicing	Harmsen et al, 2009	GGS
	PORCN_00017	F	05	c.407C>T	p.Ser136Phe	missense	Bornholdt et al, 2009 GGS
	PORCN_00018	F	05	c.479dupA	p.Tyr160X	nonsense	Wang et al, 2007	GGS
	PORCN_00019	M	05	c.502G>A	p.Gly168Arg	missense	Bornholdt et al, 2009 GGS
		F					Maas et al, 2009
	PORCN_00020	F	05	c.509G>A	p.Trp170X	nonsense	Harmsen et al, 2009	GGS

Table 3 : Large deletions listed in the PORCN database

 3 OATL1 and TBC1D25 are synonymous ; ( ¶) minimal size of a deletion includingPORCN and EBP; (*) to be confirmed (*) deletions breakpoints characterized in Bornholdt et al, 2009; (#) the novel mutations in this paper are shaded

	DB_ID_Gender	Patient_ID	DNA change	Effect on	Other genes	Phenotype	Origin	X-inactivation	Detection	Reference
				protein	deleted				method
	PORCN_00064_F	GG26	8766 bp (ex1-4)	del PORCN	no	classical	sporadic	83/17	arrayCGH/MAPH	Bornholdt et al, 2009
	PORCN_00075_F	case report	112 kb deletion	del PORCN,	SLC38A5, FTSJ1,	mild	familial	100/0	array CGH	Houge et al, 2008
					EBP, OATL1 §	(angioma
						serpiginosum)
	PORCN_00065_F	GG25	~ 20 kb (min.) ¶	del PORCN	several including	classical	sporadic	98/2	arrayCGH/MAPH	Bornholdt et al, 2009
					EBP
	PORCN_00066_F	GG36	~ 20 kb (min.) ¶	del PORCN	several including	classical	sporadic	100/0	MAPH	Bornholdt et al, 2009
					EBP
	PORCN_00067_F	GG37	~ 20 kb (min.) ¶	del PORCN	several including	classical	familial	100/0	MAPH	Bornholdt et al, 2009
					EBP
	PORCN_00068_F	GG1	137.4 kb deletion	del PORCN	SLC38A5, FTSJ1,	mild	familial	95/5	arrayCGH/MAPH	Grzeschik et al, 2007*
					EBP, TBC1D25
	PORCN_00073_F	FIN2	150 kb deletion (max.)	del PORCN	SLC38A5, FTSJ1,	classical	de novo	non informative	qPCR/arrayCGH	Froyen et al, 2009
					EBP, TBC1D25,RBM3,
					WDR13*
	PORCN_00072_F	G17	219 kb deletion	del PORCN	SLC38A5, FTSJ1,	classical	de novo	100/0	qPCR/arrayCGH/	Wang et al, 2007
					EBP, OATL1, RBM3,
					WDR13
	PORCN_00071_F	G43	~219kb deletion	del PORCN	SLC38A5, FTSJ1	non documented	not available	100/0	qPCR/arrayCGH/	Wang et al, 2007
					EBP, OATL1, RBM3,				FISH
					WDR13
	PORCN_00069_F	GG2	364.8 kb deletion	del PORCN	SSX1, SSX9,	classical	familial	99/1	arrayCGH/MAPH	Grzeschik et al, 2007*
					SSX3,SSX4,SSX4B,
					SLC38A5,FTSJ1, EBP
	PORCN_00070_F	GG11	377.5 kb deletion	del PORCN	SSX9, SSX3,SS4,	classical	familial	97/3	arrayCGH/MAPH	Grzeschik et al, 2007*
					SSX4B, SLC38A5,
					FTSJ1, EBP, TBC1D25,
					RBM3
	PORCN_00078_F	AMC_11	502 kb deletion	del PORCN	several	mild	familial	99/0	arrayCGH	this report	#
	___________________________________________________________________________________________________________________________________________________________________________
	( §)

John Wiley & Sons, Inc.Human Mutation

* Nomenclature as in the original description; ± HGVS Nomenclature p.Met1?; ¶ ? indicates that the effect on protein in not predictable: either no protein is produced or the translation initiation site moves up-or downstream; # the novel mutations in this paper are shaded; § previously described variant identified in an