**2. Thermoplastic starch preparation**

The gelatinization of starch is a process that permits the release of starch macromolecules from granules. It can be carried out by exposing starch granules to heat and shear in the presence of moisture. In the gelatinization of starch during extrusion, it is important to have strict control of the energy applied and the moisture content. The gelatinization process is depicted in Figure 1. The application of excessive heat and shear, such as that observed during extrusion processing of starch at low moisture content, leads to its thermo-mechanical degradation (Gomez & Aguilera, 1983, 1984, Lai & Kokini, 1991). Products of starch degradation are mainly dextrin, and in more extreme cases oligomer and sugar (Gomez & Aguilera, 1983, 1984). Once starch granules are disrupted, the resulting gelatinized starch (GS) can be mixed with a

Melt Blending with Thermoplastic Starch 5

processing temperature far below the processing conditions of most synthetic polymers, i.e. >150C, in order to avoid water vapor bubbles into TPS extrudates (Souza & Andrade, 2002; Farhat et al., 2003; Ma et al, 2006; Chaudhary, 2010). The development of an extruder configuration having a venting zone after both starch gelatinization and plasticization processes were accomplished and before exiting from the die allowed the preparation of

The rheological and thermal properties of water-free TPS materials having high glycerol contents (29, 36 and 40%) were evaluated by DSC analysis and rheological measurements in shear and oscillatory modes (Rodriguez-Gonzalez et al., 2004). TPS materials were labeled according to their glycerol content. Hence, TPS29,33, TPS36 and TPS40 have 29, 33, 36 and

Fig. 2. DSC thermograms of TPS samples conditioned for 24h at 0% R.H. The glycerol

As previously mentioned, TPS materials prepared in this work are almost water-free starchglycerol systems. Compared with previous work, TPS materials prepared in this work are binary systems which allow a more straightforward evaluation of the effect of glycerol on the thermal transitions of starch. DSC analysis of TPS shows a thermal transition below ambient temperature that decreases as glycerol content increases (Figure 2). On the other hand, no thermal transitions are observed between 25 and 200ºC (not shown). The Tg of TPS decreases from –45 to –56°C as glycerol content increases from 29% to 40%. Van Soest et al. have reported the Tg of extruded TPS materials containing a starch/water/glycerol ratio of 100:27:5 of +59°C (Van Soest et al., 1996). Forssell et al. (1997) studied the thermal transition of TPS materials prepared in a melt mixer as a function of glycerol and water content. Depending upon the composition, TPS materials presented one or two thermal transitions. In that work, at the lowest water content (ca. 1%) the upper transition of TPS decreases from

content in TPS is 40, 36 and 29% from the top to the bottom.

water-free TPS (Favis et al., 2001; Favis et al., 2003, Favis et al., 2005).

**3. Rheological and thermal properties of water-free TPS**

40% of glycerol.

suitable plasticizer to reduce its melting temperature and improve its processabillity. This material is known as thermoplastic starch (TPS).

Fig. 1. Schematic representation of starch gelatinization and plasticization processes during extrusion.

Water is a good plasticizer for TPS but its use leads to a high dependence of final properties to environmental conditions of humidity. Utilization of plasticizers other than water helps to stabilize the properties of TPS.   In the case of melt mixing processes, starch, plasticizer and water have been fed as dry blends (Ma & Yu, 2004a; Ma & Yu, 2004b; Da Roz et al., 2006; Ma et al, 2006; Chaudhary, 2010) or slurries (Rodriguez-Gonzalez et al., 2003a; Rodriguez-Gonzalez et al., 2003b; Rodriguez-Gonzalez et al., 2004; Tena-Salcido et al., 2008; Mendez-Hernandez et al., 2011). In some cases, TPS materials prepared by melt mixing have a significant water content which limits the

suitable plasticizer to reduce its melting temperature and improve its processabillity. This

Fig. 1. Schematic representation of starch gelatinization and plasticization processes during

Water is a good plasticizer for TPS but its use leads to a high dependence of final properties to environmental conditions of humidity. Utilization of plasticizers other than water helps to stabilize the properties of TPS.   In the case of melt mixing processes, starch, plasticizer and water have been fed as dry blends (Ma & Yu, 2004a; Ma & Yu, 2004b; Da Roz et al., 2006; Ma et al, 2006; Chaudhary, 2010) or slurries (Rodriguez-Gonzalez et al., 2003a; Rodriguez-Gonzalez et al., 2003b; Rodriguez-Gonzalez et al., 2004; Tena-Salcido et al., 2008; Mendez-Hernandez et al., 2011). In some cases, TPS materials prepared by melt mixing have a significant water content which limits the

material is known as thermoplastic starch (TPS).

extrusion.

processing temperature far below the processing conditions of most synthetic polymers, i.e. >150C, in order to avoid water vapor bubbles into TPS extrudates (Souza & Andrade, 2002; Farhat et al., 2003; Ma et al, 2006; Chaudhary, 2010). The development of an extruder configuration having a venting zone after both starch gelatinization and plasticization processes were accomplished and before exiting from the die allowed the preparation of water-free TPS (Favis et al., 2001; Favis et al., 2003, Favis et al., 2005).

#### **3. Rheological and thermal properties of water-free TPS**

The rheological and thermal properties of water-free TPS materials having high glycerol contents (29, 36 and 40%) were evaluated by DSC analysis and rheological measurements in shear and oscillatory modes (Rodriguez-Gonzalez et al., 2004). TPS materials were labeled according to their glycerol content. Hence, TPS29,33, TPS36 and TPS40 have 29, 33, 36 and 40% of glycerol.

Fig. 2. DSC thermograms of TPS samples conditioned for 24h at 0% R.H. The glycerol content in TPS is 40, 36 and 29% from the top to the bottom.

As previously mentioned, TPS materials prepared in this work are almost water-free starchglycerol systems. Compared with previous work, TPS materials prepared in this work are binary systems which allow a more straightforward evaluation of the effect of glycerol on the thermal transitions of starch. DSC analysis of TPS shows a thermal transition below ambient temperature that decreases as glycerol content increases (Figure 2). On the other hand, no thermal transitions are observed between 25 and 200ºC (not shown). The Tg of TPS decreases from –45 to –56°C as glycerol content increases from 29% to 40%. Van Soest et al. have reported the Tg of extruded TPS materials containing a starch/water/glycerol ratio of 100:27:5 of +59°C (Van Soest et al., 1996). Forssell et al. (1997) studied the thermal transition of TPS materials prepared in a melt mixer as a function of glycerol and water content. Depending upon the composition, TPS materials presented one or two thermal transitions. In that work, at the lowest water content (ca. 1%) the upper transition of TPS decreases from

Melt Blending with Thermoplastic Starch 7

Fig. 4. Effect of glycerol content on (a) elastic modulus (G') and (b) loss modulus (G") of TPS

materials evaluated at 150ºC

145 to 70°C as the glycerol content is increased from 14 to 29% while only TPS compounded with 29 and 39% glycerol showed lower transitions both at -50°C. The upper transition was attributed to starch-rich phase while the lower transition was related to a starch-poor phase. Lourdin and coworkers prepared TPS cast films by mixing starch with different amounts of water and glycerol (Lourdin et al., 1997a; Lourdin et al., 1997b). Films having around 13% water content showed a reduction of Tg from 90 to 0°C when glycerol content increased from 0 to 24% (Lourdin et al., 1997a). In that case they observed a glassy to rubbery transition of TPS at around 15% glycerol. In a further paper, they compared the Tg of TPS films having around 11% water with respect to glycerol content and they found that Tg decreased from 126 to 28°C when glycerol content was increased from 0 to 40% (Lourdin et al., 1997b). Discrepancies in Tg values as a function of glycerol content can be related, as mentioned by Kalichevsky to the mixing history during TPS preparation (Kalichevsky et al., 1993).

During on-line measurements, TPS extrudates did not present bubbles due to the almost absence of water. The pressure readings of TPS36 and TPS40 at 150ºC were quite regular while those of TPS29 were mostly irregular. For this reason only TPS36 and TPS40 were evaluated. As observed by other authors (Aichholzer and Fritz, 1998; Della Valle et al., 1992; Lai and Kokini, 1990; Senouci and Smith, 1988; Willett et al., 1995; Willett et al., 1998), the viscosity () of both TPS and PE1 melts display a power-law (shear thinning) behavior at the shear rate ( ) interval developed over die extrusion conditions (Figure 3). The of TPS materials depends on the plasticizer content. An increment of glycerol content from 36% to 40% results in a reduction of 20% of of TPS36 (at ~ 130 s-1).

Fig. 3. Comparison of the viscosity of TPS40, TPS36 and PE1 measured on-line in the TSE at 150ºC.

145 to 70°C as the glycerol content is increased from 14 to 29% while only TPS compounded with 29 and 39% glycerol showed lower transitions both at -50°C. The upper transition was attributed to starch-rich phase while the lower transition was related to a starch-poor phase. Lourdin and coworkers prepared TPS cast films by mixing starch with different amounts of water and glycerol (Lourdin et al., 1997a; Lourdin et al., 1997b). Films having around 13% water content showed a reduction of Tg from 90 to 0°C when glycerol content increased from 0 to 24% (Lourdin et al., 1997a). In that case they observed a glassy to rubbery transition of TPS at around 15% glycerol. In a further paper, they compared the Tg of TPS films having around 11% water with respect to glycerol content and they found that Tg decreased from 126 to 28°C when glycerol content was increased from 0 to 40% (Lourdin et al., 1997b). Discrepancies in Tg values as a function of glycerol content can be related, as mentioned by Kalichevsky to the

During on-line measurements, TPS extrudates did not present bubbles due to the almost absence of water. The pressure readings of TPS36 and TPS40 at 150ºC were quite regular while those of TPS29 were mostly irregular. For this reason only TPS36 and TPS40 were evaluated. As observed by other authors (Aichholzer and Fritz, 1998; Della Valle et al., 1992; Lai and Kokini, 1990; Senouci and Smith, 1988; Willett et al., 1995; Willett et al., 1998), the viscosity () of both TPS and PE1 melts display a power-law (shear thinning) behavior at the

materials depends on the plasticizer content. An increment of glycerol content from 36% to

Fig. 3. Comparison of the viscosity of TPS40, TPS36 and PE1 measured on-line in the TSE at

) interval developed over die extrusion conditions (Figure 3). The of TPS

~ 130 s-1).

mixing history during TPS preparation (Kalichevsky et al., 1993).

40% results in a reduction of 20% of of TPS36 (at

shear rate (

150ºC.

Fig. 4. Effect of glycerol content on (a) elastic modulus (G') and (b) loss modulus (G") of TPS materials evaluated at 150ºC

Melt Blending with Thermoplastic Starch 9

Blending TPS with synthetic polymers have shown the typical characteristics of immiscible polymer blends (St-Pierre et al, 1997). The melt blending of TPS with synthetic polymers has given place to a series of scientific and technologic developments. Such works differed in the mixing protocol and the type of additives used. Some authors proposed the use of two steps for the preparation of TPS-based blends (Aburto et al., 1997, Bikiaris et al., 1997a, 1997b, 1998, Prinos et al., 1998, Averous et al., 2000a, 2000b, 2001a, 2001b, Martin & Averous, 2001) while other preferred just one-step processes (Dehennau & Depireux, 1993, St-Pierre et al., 1997). Starch-based blends prepared in two steps are generally characterized for the preparation of TPS in a separated extrusion step. St-Pierre and coworkers presented a one-step blending process for TPS-based polymer blends (St-Pierre et al., 1997). They developed an extrusion system combining a TSE with a singlescrew extruder (SSE). TPS was prepared in the SSE, and then it was blended with LDPE in the last sections of the TSE. Using such an extrusion system, they demonstrated experimentally that a certain morphological control of PE/TPS blends could be achieved by varying the TPS concentration from 0 to 22 wt%. Those blends showed an unusual

Fig. 5. Schematic representation of the one-step extrusion system designed for the melt

An improved approach for LDPE/TPS blends in a one-step process was developed by Rodriguez-Gonzalez and coworkers (Rodriguez-Gonzalez et al., 2003b). It consisted of an extrusion system equipped with a single-screw extruder, from which molten LDPE is fed to the middle section of a twin-screw extruder. Suspensions of starch, glycerol and water were

**4. Blending with polyethylene**

high level of ductility.

blending of LDPE with water-free TPS.

TPS exhibits the rheological behavior of a typical gel as characterized by a storage modulus (G', Figure 4a) larger than the loss modulus (G", Figure 4b) and with both moduli largely independent of frequency over the amplitude of the experimental window (Ross-Murphy, 1995). This behavior is produced by the presence of an elastic network embedded in a softer matrix. The rigidity in those regions can be produced by chemical or physical crosslinking. The structure of the elastic network has been related to the crystallinity derived from the complexation reaction between amylose and lipids (Conde-Petit & Escher, 1995; Della Valle et al., 1998) and the physical entanglement of the high molecular weight polysaccharides (Della Valle et al., 1998; Ruch and Fritz, 2000).

As expected, the augmentation of the glycerol content in TPS results in a reduction of both G' and G". However, the trend in the modulus curves was nearly the same, regardless of the glycerol content. From the study of low-concentration starch dispersions, Conde-Petit and Escher (1995) showed that the formation of amylose-emulsifier complexes modifies the viscoelastic response of potato starch dispersions. Crystalline regions produced during the amylose-emulsifier complexation form an elastic network, which is responsible for the liquid-like to solid-like viscoelastic modification. From the similarity of the trend of the G' curves shown in Figure 4a, it can be inferred in this work that glycerol variation does not affect the nature of the hypothetical crystalline elastic network, it just plasticizes the amorphous fraction of starch.

The study of the viscoelasticity of starch-based materials has mainly focused on concentrated gels and dispersions ( 5% starch). In this work, the viscoelastic behavior of water-free TPS at high glycerol contents has been evaluated at 150°C. G' decreases as glycerol content increases and the changes are similar at both low and high frequencies. Della Valle and co-workers also studied the behavior of a water-free TPS at 150°C and found that the decrease of G' with glycerol content was dependent on frequency (Della Valle et al., 1998). However, that material was obtained by subjecting the TPS to a separate drying step, a process which can induce structural changes in the starch. The proportional reduction of G' as a function of glycerol content observed in this work is similar to that observed in starch gel systems (Kulicke et al., 1996). Figure 6a shows that the reduction of the glycerol content from 40% to 33% results in a quasi-linear increment of G', while the reduction from 33% to 29% glycerol produces a larger variation in G'. In the case of the elastic modulus of polymer composites, percolation theory explains the non-linearity produced by the phase inversion effect at high filler content (Willett, 1994). The limit of glycerol plasticization that produces the non-linearity observed in the G' of TPS at a concentration around 30% glycerol can be explained in a similar way. TPS can be considered as a homogeneous system composed of a hard elastic network and soft amorphous regions. Amylose complex crystallites, highly entangled starch molecules, poorly plasticized starch-rich sites, or a combination of them could compose the hard elastic network. Soft amorphous regions could be composed of well-plasticized glycerol-rich starch. Even though the elastic network is present at 33% glycerol, the soft amorphous regions dominate the viscoelastic response. Increasing glycerol content, beyond this concentration, produces a relatively small reduction in the rheological parameters. On the other hand, below 30% glycerol the phase inversion of a soft to a hard matrix occurs resulting in the domination of the viscoelastic response by the hard elastic network, which is in good agreement with percolation theory. That suggests a glycerol plasticization threshold at a concentration around 30%.

TPS exhibits the rheological behavior of a typical gel as characterized by a storage modulus (G', Figure 4a) larger than the loss modulus (G", Figure 4b) and with both moduli largely independent of frequency over the amplitude of the experimental window (Ross-Murphy, 1995). This behavior is produced by the presence of an elastic network embedded in a softer matrix. The rigidity in those regions can be produced by chemical or physical crosslinking. The structure of the elastic network has been related to the crystallinity derived from the complexation reaction between amylose and lipids (Conde-Petit & Escher, 1995; Della Valle et al., 1998) and the physical entanglement of the high molecular weight polysaccharides

As expected, the augmentation of the glycerol content in TPS results in a reduction of both G' and G". However, the trend in the modulus curves was nearly the same, regardless of the glycerol content. From the study of low-concentration starch dispersions, Conde-Petit and Escher (1995) showed that the formation of amylose-emulsifier complexes modifies the viscoelastic response of potato starch dispersions. Crystalline regions produced during the amylose-emulsifier complexation form an elastic network, which is responsible for the liquid-like to solid-like viscoelastic modification. From the similarity of the trend of the G' curves shown in Figure 4a, it can be inferred in this work that glycerol variation does not affect the nature of the hypothetical crystalline elastic network, it just plasticizes the

The study of the viscoelasticity of starch-based materials has mainly focused on concentrated gels and dispersions ( 5% starch). In this work, the viscoelastic behavior of water-free TPS at high glycerol contents has been evaluated at 150°C. G' decreases as glycerol content increases and the changes are similar at both low and high frequencies. Della Valle and co-workers also studied the behavior of a water-free TPS at 150°C and found that the decrease of G' with glycerol content was dependent on frequency (Della Valle et al., 1998). However, that material was obtained by subjecting the TPS to a separate drying step, a process which can induce structural changes in the starch. The proportional reduction of G' as a function of glycerol content observed in this work is similar to that observed in starch gel systems (Kulicke et al., 1996). Figure 6a shows that the reduction of the glycerol content from 40% to 33% results in a quasi-linear increment of G', while the reduction from 33% to 29% glycerol produces a larger variation in G'. In the case of the elastic modulus of polymer composites, percolation theory explains the non-linearity produced by the phase inversion effect at high filler content (Willett, 1994). The limit of glycerol plasticization that produces the non-linearity observed in the G' of TPS at a concentration around 30% glycerol can be explained in a similar way. TPS can be considered as a homogeneous system composed of a hard elastic network and soft amorphous regions. Amylose complex crystallites, highly entangled starch molecules, poorly plasticized starch-rich sites, or a combination of them could compose the hard elastic network. Soft amorphous regions could be composed of well-plasticized glycerol-rich starch. Even though the elastic network is present at 33% glycerol, the soft amorphous regions dominate the viscoelastic response. Increasing glycerol content, beyond this concentration, produces a relatively small reduction in the rheological parameters. On the other hand, below 30% glycerol the phase inversion of a soft to a hard matrix occurs resulting in the domination of the viscoelastic response by the hard elastic network, which is in good agreement with percolation theory. That suggests a

glycerol plasticization threshold at a concentration around 30%.

(Della Valle et al., 1998; Ruch and Fritz, 2000).

amorphous fraction of starch.

### **4. Blending with polyethylene**

Blending TPS with synthetic polymers have shown the typical characteristics of immiscible polymer blends (St-Pierre et al, 1997). The melt blending of TPS with synthetic polymers has given place to a series of scientific and technologic developments. Such works differed in the mixing protocol and the type of additives used. Some authors proposed the use of two steps for the preparation of TPS-based blends (Aburto et al., 1997, Bikiaris et al., 1997a, 1997b, 1998, Prinos et al., 1998, Averous et al., 2000a, 2000b, 2001a, 2001b, Martin & Averous, 2001) while other preferred just one-step processes (Dehennau & Depireux, 1993, St-Pierre et al., 1997). Starch-based blends prepared in two steps are generally characterized for the preparation of TPS in a separated extrusion step. St-Pierre and coworkers presented a one-step blending process for TPS-based polymer blends (St-Pierre et al., 1997). They developed an extrusion system combining a TSE with a singlescrew extruder (SSE). TPS was prepared in the SSE, and then it was blended with LDPE in the last sections of the TSE. Using such an extrusion system, they demonstrated experimentally that a certain morphological control of PE/TPS blends could be achieved by varying the TPS concentration from 0 to 22 wt%. Those blends showed an unusual high level of ductility.

Fig. 5. Schematic representation of the one-step extrusion system designed for the melt blending of LDPE with water-free TPS.

An improved approach for LDPE/TPS blends in a one-step process was developed by Rodriguez-Gonzalez and coworkers (Rodriguez-Gonzalez et al., 2003b). It consisted of an extrusion system equipped with a single-screw extruder, from which molten LDPE is fed to the middle section of a twin-screw extruder. Suspensions of starch, glycerol and water were

Melt Blending with Thermoplastic Starch 11

Fig. 6. Effect of glycerol content and LDPE viscosity on the morphology of microtomed PE/TPS (70/30) blends. PE1/TPS blends: a) 40% glycerol, b) 36% glycerol, and c) 29% glycerol. d) PE2/TPS at 29% glycerol content. The black bar below the micrographs

represents 10m.

fed to the hopper of the twin-screw extruder and, as described in section 3, water-free TPS having 29, 36 and 40% glycerol (TPS29, TPS36 and TPS40, respectively) were prepared and melt blended with the LDPE as depicted in Figure 5. In order to evaluate the effect of PE and TPS viscosities on the morphology of LDPE/TPS blends two commercial LDPE resins, LDPE2040 (PE1, MFI = 12g/10min) and LDPE2049 (PE2, MFI = 20g/10min), and the three TPS were used.


3.2.2. Lead Pyrochlore (Naples Yellow)

A second important outcome of this study was the detection of at least two types of Naples yellow lead pyrochlore pigments in the yellow and green painted enamels of the porcelains. Naples yellow refers to a variety of synthetic lead antimonate pigments with a pyrochlore structure, prepared by mixing lead oxide with various compounds, including mainly antimony and tin, in the presence of various fluxing agents [1,3,12,13,18,47–54]. This pigment, in the form of Sb- or Sn-rich types, has been used as a glass and glaze pigment since antiquity [55–57] and as a painting pigment after the Quattrocento/Renaissance [53,54]. Possible variations of the pigment might also include the addition of zinc, iron and silicon elements [14,18,49,50]. The adjustment of the proportions of these compounds at varying firing temperatures allows for the production of modified forms of the pigment displaying different hues. The composition of Naples yellow type pigments is therefore nonstoichiometric with the incorporation of various cations into the lead pyrochlore structure (Pb2−xM'xM2−YM"YO7−δ with M,M" = Sb, Sn, Fe, Si, Zn; M' = RE). This phase is built with two sublattices, one of (big) Pb2+ cations, the second of (small, covalent-bonded) cations forming tetrahedral and octahedral entities sharing some oxygen atoms. During the firing process of the enamels, composition of the pigment used may be further affected with other species present in the enamel matrix (Fe, Si, Sn, etc.) [14,18,50,58,59]. Some of these incorporating elements may also have different speciations (e.g., Sn2+ or Sn4+, Sb3+ or Sb5+, Fe2+ or Fe3+), and furthermore, the oxygen nonstoichiometry very much depends on the conditions of the firing process (reducing/oxidizing conditions).

In practice, the lead pyrochlore pigment can be prepared as a powder in advance and then mixed with the glass/glaze powder before deposition, the so-called "*anime*" and "*corpo*" recipes [57,60,61]. Alternatively, all the ingredients can be mixed together to obtain the yellow glass/glaze powder. In the first method, the grains of lead pyrochlore are rather big (>>µm), while in the other, the homogeneous nucleation on cooling gives rise to small precipitates (<a few µm). With a high-magnification microscope objective, a single pigment grain could be analyzed, and the Raman spectrum obtained was strong without a background, and almost no signature of the glassy matrix was recorded (see e.g., Figure 7A—TH457 yellow; see also in ref. [34]). In the case of nucleation, both the signature of lead pyrochlore precipitates and glass were recorded together with a significant background (e.g., Figure 6D(c,c')).

In the last decades, several Raman spectrometry studies performed on various pottery glazes, glasses and enamels [1–3,11–18,47–52,57–59] as well as oil paintings [53–56] allowed for the identification of Naples yellow lead pyrochlore pigments. In general, these pigments have a very characteristic Raman signature, which basically includes the strong stretching mode peaks of Pb ions at the low-wavenumber region (~115–145 cm−1 ) and M/M"–O stretching modes from ~300 to 600 cm−1 . Furthermore, Sb-rich pyrochlore pigment is mainly distinguished by strong peaks at ~110–130 and ~505 cm−1 , while Sn-rich-type pigment exhibits stronger peaks at ~130–135, ~335 and 450 cm−1 (the Sn–O stretching mode) with the disappearance of the ~505 cm−1 peak [10,13,18,47–52]. Zn-containing mixed lead pyrochlores exhibit a medium 450–480 cm−1 component [18]. Although Sn-rich lead pyrochlore was observed in most of the yellow and green enameled areas (Figure 6B(c) (R1048), Figure 6D(c,c') (R1135), Figure 9A(a) (R1006), Figure 9B(a,a') (SN284), Figure 10A(b,b') (TH487), Figure 11A(a) (R1056) and Figure 13B(c,c') (F1341C)), Sb-rich compositions were clearly observed in the yellow enamels of the TH457 bottle from the Thiers Collection (Figures 3 and 7A) and the R1175 plate with an English family's coat of arms (Figures 2 and 8 A(a,a')). Mixed lead pyrochlores were also observed in some cases (Figure 7B(a,a') (F1371C) and Figure 12 B(b,b',b") (F1429C)). As a second piece of proof, the XRF spectrum recorded on the yellow enamel of TH457 showed both Sn and Sb signals (Figures 14 and 15).

signals (Figures 14 and 15).

3.2.3. Red to Violet Colors

*Heritage* **2020**, *3* FOR PEER REVIEW 15 of 27

mixed lead pyrochlores exhibit a medium 450–480 cm−1 component [18]. Although Sn-rich lead pyrochlore was observed in most of the yellow and green enameled areas (Figure 6B(c) (R1048), Figure 6D(c,c') (R1135), Figure 9A(a) (R1006), Figure 9B(a,a') (SN284), Figure 10A(b,b') (TH487), Figure 11A(a) (R1056) and Figure 13B(c,c') (F1341C)), Sb-rich compositions were clearly observed in the yellow enamels of the TH457 bottle from the Thiers Collection (Figures 3 and 7A) and the R1175 plate with an English family's coat of arms (Figures 2 and 8 A(a,a')). Mixed lead pyrochlores were

**Figure 14.** Representative portable XRF (pXRF) spectra recorded on the TH457 bottle for the glaze and different painted enamels. **Figure 14.** Representative portable XRF (pXRF) spectra recorded on the TH457 bottle for the glaze and different painted enamels. *Heritage* **2020**, *3* FOR PEER REVIEW 16 of 27

**Figure 15.** Representative pXRF spectra recorded on the TH457 bottle for pink and black enameled areas; bottom: zoom in on the energy window characteristics of Sn and Sb elements for different areas. **Figure 15.** Representative pXRF spectra recorded on the TH457 bottle for pink and black enameled areas; bottom: zoom in on the energy window characteristics of Sn and Sb elements for different areas.

8A(c,d), orange and red; and Figure 8B(c), red), the use of both methods was observed. Hematite has a well-defined Raman signature, with a strong (ca. 1305–1310 cm−1) mode (resonance signal of magnon mode under green laser excitation) and narrow peaks between 200 and 600 cm−1 [62,63] (see e.g., Figures 6D(b,b',b''), 7A, 8A(c), 8B(c) and 12C(b,b')). The magnon mode is very sensitive to the particle size of hematite [63] and the oxygen stoichiometry [62], with small particles giving an orangered color [29]. Broadening of hematite peaks indicates a partial substitution of iron with other

A specific Raman feature referred to the use of gold nanoparticles dispersed in the glassy matrix of the enamel (this type of enamel is at the origin of the label "*Famille rose*" [15,19], with a characteristic broad fluorescence background peaking at ~500–600 cm−1 under a green laser, corresponding to ~500 nm on the absolute scale [14,18]; see Figure 6C(c) (R1041), Figure 7A (TH457), Figure 8A(d) (R1175), Figure 9B(c) (SN284), Figure 10A(a,a'),B(d) (TH487), Figure 12C(a) (F1429C) and Figure 13C(a) (F1341C)). This method was first experimented with in 17th-century France by the glassmaker Bernard Perrot and then some years later by Johann Kunckel in Germany and certainly by others in Italy before [16,18,64–67]. The presence of gold was also confirmed by pXRF analysis (see arrow on Figure 15—pink, in which the small Au Lα peak is obvious), with a hardly visible peak well-identified by data fitting. In addition, it is important to note that a very small band characteristic of the As–O stretching band at ca. 820 cm−1 could be detected along with the fluorescence background characteristic of the use of colloidal gold (seen clearly in Figure 10A(a,a') (TH487) and less intensely in Figure 9B(c) (SN284) and Figure 13C(a) (F1341C)). This band corresponds to the use of Perrot's preparation route for obtaining the colloidal gold precipitate using an arsenic salt, which is different

elements, possibly Ti and Al coming from the iron sources [29].

The last important characteristic of the painted enameled porcelains analyzed was the presence


**3. Single cell analysis based on an E‐SEM nanorobotic manipulation system**

Single cells analysis needs to be investigated through the micro/nano scale techniques based on the local environmental measurements and controls. We developed the Environmental‐ SEM (E‐SEM) nanorobotic manipulation system to manipulate and control the local environ‐ ments for biological samples in nano scale (Figure 2). It realized that direct observation and manipulation of water‐containing biological samples under nanometer high resolution imaging.

**Figure 2.** Single Cell Nano-surgery System with various nano-tools

microscopes (SEMs) and transmission electron microscopes (TEMs). For example, M. F. Yu et. al presented the tensile strength of individual CNTs inside a SEM [18]. Howeverthe resolution of SEM, generally ~1 nm resolution, is approximately one order in magnitude lower than that of a TEM. High resolution and transmission image of TEMs are useful for measurement and evaluation of nano‐scale objects. Kizuka et.al proposed the manipulation holder inside high‐ resolution transmission electron microscope (HR‐TEM). The manipulator was specially

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

However, the specimen chamber and observation area of TEM are too narrow to contain manipulators with complex functions. Hence, special sample preparation techniques are also needed. We proposed a hybrid nanorobotic manipulation system which is integrated TEM and SEM nanorobotic manipulators as core system for the Nanolaboratory [20, 21]. The strategy is named as hybrid nanomanipulation so as to differentiate it from those with only an exchange‐ able specimen holder. The most important feature of the manipulatoris that it contains several passive DOFs, which makes it possible to perform relatively complex manipulations whereas to keep compact volume to be installed inside the narrow vacuum chamber of a TEM [22].

Recently single cells analysis has been much more attentions because of the progress of the micro/nano scale techniques on the local environmental measurements and controls [23]. Under conventional SEMs and TEMs, the sample chambers of these electron microscopes are set under the high vacuum (HV) to reduce the disturbance of electron beam for observation. To observe water‐containing samples, for example bio‐cells, the appropriate drying and dying treatments are needed before observations. Hence, direct observations of water‐containing

On the other hand, the environmental‐SEM (E‐SEM) can be realized the direct observation of water‐containing samples with nanometer high resolution by specially built secondly electron detector [24]. The evaporation of water is controlled by the sample temperature (~0 ‐ ~40 °C) and sample chamber pressure (10 ‐ 2600 Pa). The unique characteristic of the E‐SEM is the direct observation of the hydroscopic samples with non‐drying treatment. Hence, the nano‐ manipulation inside the E‐SEM is considered to be an effective tool for a water‐containing

**3. Single cell analysis based on an E‐SEM nanorobotic manipulation**

Single cells analysis needs to be investigated through the micro/nano scale techniques based on the local environmental measurements and controls. We developed the Environmental‐ SEM (E‐SEM) nanorobotic manipulation system to manipulate and control the local environ‐ ments for biological samples in nano scale (Figure 2). It realized that direct observation and manipulation of water‐containing biological samples under nanometer high resolution

samples are normally quite difficult through these electron microscopes.

sample with nanometer resolution [25‐28].

**system**

imaging.

designed with atomic level positioning resolution [19].

Biomedical Engineering

356

In this chapter, the novel local stiffness evaluation, local cutting, and local extraction of biological organism are presented by micro‐nanoprobes based on the E‐SEM nanorobotic manipulation system for future cell diagnosis and surgery system.

### **3.1. Adhesion force measurement of single cell using nano‐putter**

Cell activities, such as embryogenesis, mitosis, morphogenesis, cell orientation, cell motility, and survival depend on attachment to neighboring cells and the extracellular matrix. Cellular attachment to extracellular matrices influences cell morphology, cell function, and signaling mechanisms that direct cellular proliferation and differentiation [29]. Cell‐surface interaction is important in the development of any material or device for biomedical applications, since the performance of a medical device in the body must be compatible with the surrounding tissue [30, 31]. Understanding of the cell adhesion process would benefit the development of suitable biomaterials or device for both tissue engineering and medical fields.

Cell adhesion processes are influenced by numerous parameters, such as the nature of the biomaterial and its surface characteristics (roughness, topography, chemical composition, surface wettability, surface charge and surface treatments) have been investigated. However, the effect of ambient humidity on cell adhesion has had less attention, especially at the single cell level. Understanding the adhesion force at various humidity conditions could help us to better understand the processes of cell‐directed integration during water evaporation. The understanding is also useful in controlling yeast infections at wet environment. Moreover, cell adhesion is influenced by the surface energy of substrate strongly, but the mechanism is still not clear[32]. The study of cell adhesion on substrates with different surface energy could help us to understand the adhesion mechanism better.

We presented a yeast cell adhesion force measurement performed using the nanorobotic manipulation system inside the ESEM [33, 34, 35]. Figure 3 (A) shows a typical force‐displace‐ ment curve during the single cell adhesion force measurement. Figure 3 (B) shows the initial position of the micro putter and the single cell. The micro putter was driven by the nanorobotic manipulation system. First, it was moved towards the cell until it contacted the cell (Figure 3 (C)). Then, a continuous movement was applied to the micro putter by the nanomanipulator. The micro putter beam deflected owing to the increasing pushing force. Figure 3 (D) shows the deflection of the micro putter during the adhesion force measurement. Finally, the cell was detached from its initial position under a certain force (Figure 3 (E)). The maximum force during this manipulation procedure was defined as the adhesion force.

Single yeast cell adhesion force measurement was performed at three humidity conditions, i.e. 100%, 70% and 40%. The mean adhesion force and the deviation are with 95% confidence at each humidity conditions. It demonstrates that the yeast cell adhesion forces range from 10 to 25 μN at various humidity conditions. The adhesion forces were 11.0 ± 5.1 μN, 17.4 ± 4.7 μN and 23.5 ± 6.1 μN at 100%, 70% and 40% relative humidity conditions respectively. It showed clearly that the cell adhesion was affected by the ambient humidity. The cell adhesion force is larger at low humidity than at high humidity. For example, the cell adhesion force was 23.5 μN at a humidity of 40%, which was 1.14 times largerthan the force 11.0 μN at humidity 100%.

#### **3.2. Single cell cutting using nano‐knife**

Cell cutting is an important step in cell analysis processes. For instance, it was widely used to prepare cell specimen slices for the observation of an inner structure [36]. Different to group cells analysis,research on individual cells could give accurate data ratherthan average results. Single cell analysis can help us to understand the biological processes more accurately. In‐situ single cell cutting technique could potentially benefit cell analysis, such as single cell operation and disease treatment.

Recently, a nano knife fabricated from a carbon nanotube (CNT) has been developed for the purpose of cell cutting [37]. The nano knife was designed by welding a CNT across two tungsten needles inside a scanning electron microscopy (SEM). This device can reduce the angle by which the sample is bent during cutting, due to the small diameter of the CNT. It can be seen clearly that the nano knife can leave a mark on the epon resin surface, which means it can cut very thin slices of cells. However, the bonding force between the CNT and tungsten probes by using electron beam induced deposition (EBID) method is quite small. There are still certain types of hard specimens such as bone, plants, and thick‐walled spores. The CNT based nano knife may not be able to deal with such samples, since a larger cutting force is required, especially when the sample size is large.

We presented a nano knife with a buffering beam was designed for an in‐situ single cell cutting purpose [38]. A schematic drawing of the single cell cutting using a nano knife is shown in Figure 4. The nano knife was immobilized to the nanomanipulator by using the electrical conductive tape inside the ESEM chamber. Underthe driving of the nanorobotic manipulation system, the nano knife can move towards and cut the single cell finally. The cutting force can be calculated based on the deformation of the nanokinfe's beam.

**Figure 3.** Single cell adhesion force measurement using micro putter inside ESEM.

Single Cell Nanosurgery System 359

**Figure 3.** Single cell adhesion force measurement using micro putter inside ESEM.

We presented a yeast cell adhesion force measurement performed using the nanorobotic manipulation system inside the ESEM [33, 34, 35]. Figure 3 (A) shows a typical force‐displace‐ ment curve during the single cell adhesion force measurement. Figure 3 (B) shows the initial position of the micro putter and the single cell. The micro putter was driven by the nanorobotic manipulation system. First, it was moved towards the cell until it contacted the cell (Figure 3 (C)). Then, a continuous movement was applied to the micro putter by the nanomanipulator. The micro putter beam deflected owing to the increasing pushing force. Figure 3 (D) shows the deflection of the micro putter during the adhesion force measurement. Finally, the cell was detached from its initial position under a certain force (Figure 3 (E)). The maximum force

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

Single yeast cell adhesion force measurement was performed at three humidity conditions, i.e. 100%, 70% and 40%. The mean adhesion force and the deviation are with 95% confidence at each humidity conditions. It demonstrates that the yeast cell adhesion forces range from 10 to 25 μN at various humidity conditions. The adhesion forces were 11.0 ± 5.1 μN, 17.4 ± 4.7 μN and 23.5 ± 6.1 μN at 100%, 70% and 40% relative humidity conditions respectively. It showed clearly that the cell adhesion was affected by the ambient humidity. The cell adhesion force is larger at low humidity than at high humidity. For example, the cell adhesion force was 23.5 μN at a humidity of 40%, which was 1.14 times largerthan the force 11.0 μN at humidity 100%.

Cell cutting is an important step in cell analysis processes. For instance, it was widely used to prepare cell specimen slices for the observation of an inner structure [36]. Different to group cells analysis,research on individual cells could give accurate data ratherthan average results. Single cell analysis can help us to understand the biological processes more accurately. In‐situ single cell cutting technique could potentially benefit cell analysis, such as single cell operation

Recently, a nano knife fabricated from a carbon nanotube (CNT) has been developed for the purpose of cell cutting [37]. The nano knife was designed by welding a CNT across two tungsten needles inside a scanning electron microscopy (SEM). This device can reduce the angle by which the sample is bent during cutting, due to the small diameter of the CNT. It can be seen clearly that the nano knife can leave a mark on the epon resin surface, which means it can cut very thin slices of cells. However, the bonding force between the CNT and tungsten probes by using electron beam induced deposition (EBID) method is quite small. There are still certain types of hard specimens such as bone, plants, and thick‐walled spores. The CNT based nano knife may not be able to deal with such samples, since a larger cutting force is

We presented a nano knife with a buffering beam was designed for an in‐situ single cell cutting purpose [38]. A schematic drawing of the single cell cutting using a nano knife is shown in Figure 4. The nano knife was immobilized to the nanomanipulator by using the electrical conductive tape inside the ESEM chamber. Underthe driving of the nanorobotic manipulation system, the nano knife can move towards and cut the single cell finally. The cutting force can

during this manipulation procedure was defined as the adhesion force.

**3.2. Single cell cutting using nano‐knife**

required, especially when the sample size is large.

be calculated based on the deformation of the nanokinfe's beam.

and disease treatment.

Biomedical Engineering

358

The in situ single cell cutting experiment was performed using these three nano knives. Figure 5(A) shows the initialposition ofthe nano knife anda single cell. Figure 5(B) shows the touching between the nano knife tip and the single cell. The deformation of the nano knife beam and the single cell during the cutting is shown in Figure 5(C). The deformation of the beam can be measured from the ESEM image directly using image analysis software. Therefore, the cutting force can be calculated based on Hooke's law. The separated single cell after cutting is shown in Figure 5(D). The sample slice angle can be measured from the ESEM image directly as well. Figure 5(E) shows the single cell cutting image using the 25o knife. Figure 5(F) shows the sample slice angle after cutting. The images of single cell cutting and sample slice angle after cutting using 45o knife are shown in Figure 5 (G) and Figure 5 (H).

**4. Conclusion**

one of the model organisms [40, 41]

**Acknowledgements**

**Author details**

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TRENDS in Pharmacological Sciences 2005;26 265‐273.

This chapter presents the single cell nanosurgery system based on nanomanipulation techni‐ ques.The micro‐nano tools have been proposed to investigate single cell analysis to manipulate and control the local environment in micro‐nano scale. The E‐SEM nanomanipulation system was constructed to realize the local stiffness evaluation, local cutting, and local extraction of biological organism in nano‐meter scale. The adhesion force measurement was presented by micro‐putter for single cells. The single cell cutting was also described using nano‐knife. As future direction, the multiple micro‐nanotools are used continuously depending on the purposes by exchanging machinery system (NTExS: Nanotool Exchanger System) [39]. We are investigating on the nanoinjection applications for the Caenorhabditis elegans (C. elegans) as

Single Cell Nanosurgery System 361

The authors are grateful to Prof. T. Inada N. Uozumi for providing with W303 yeast cells. This work was partially supported by a Grant‐in‐Aid for Scientific Research from the Ministry of

Education, Culture, Sports, Science and Technology of Japan.

Toshio Fukuda, Masahiro Nakajima, Yajing Shen and Masaru Kojima

Department of Micro‐Nano Systems Engineering, Nagoya University, Nagoya, Japan

[1] Fields S, Johnston M. Cell biology: Whither model organism research? Science

[2] Fox M. A. The case for animal experimentation: An evolutionary and ethical perspec‐

[3] Botstein D, Chervitz S. A, Cherry J. M. Yeast as a model organism. Science 1997;277

[4] Foury F. Human genetic diseases ? a cross‐talk between man and yeast. Gene

[5] Mager W. H, Winderickx J. Yeast as a model for medical and medicinal research.

**Figure 4.** Schematic drawing of the single cell cutting experiment using nano knife inside ESEM.

**Figure 5.** Single cell cutting using nano knife inside ESEM.


**1. Introduction**

Our brain is an extraordinary microsome and has been completely shrouded in mystery. However, its mystery has been just a little bit by bit solved owing to recent advances in experimental technologies and tremendous development of computers. Many people can simply say "brain," but it is a general term for a collection of six main regions, that is, cerebrum, diencephalon, midbrain, cerebellum, pons, and medulla oblongata. The brain is an organ that

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

takes the central role in advanced information processing, such as visual, auditory, speech or language faculties, motion control, recognition, emotion, and so on. According to advances of experiment and computer technology, the research of brain science or neuroscience has been made not only in the fields of medicine, biology, biochemistry, pharmacology, and psycholo‐ gy but also in the field of engineering.

The present-day computers have outstanding processing capacity. For example, they can find the data that satisfy some requirements among huge quantities of data (database) or can calculate over five trillion figures of pi(π). Therefore, many people are inclined to think that our brain will be able to be replaced by computer in near future. Surely, computers excel at processing of digitized data and processing by following a standard algorithm. However, it can hardly execute processing, such as recognition of ambiguity figures (such as illusionism) or inference based on imperfect information, which our brain can instantaneously carry out. Reason for this comes from differences in ways of information processing of the computer and our brain. The current computers, called von Neumann computer, are grounded in sequential processing by using central processing units (CPUs) and memory storages, while on the other hand, our brain bases on parallel and distributed processing through neural networks whose components are neurons.

There exist tens of billions of neurons in our brain, which build neural networks in complicated arrangement. All information processing in the brain is accomplished by neural activity in the form of neural oscillations that cause cortical oscillations (delta, theta, alpha, beta, or gamma oscillation). In order to clarify the mechanisms of advanced information processing in the brain, such as learning and memory, it is necessary to understand functions and features of neurons and neural networks. Although the current progress in experiment technology and measuring system is remarkable, only experiments by themselves cannot uncover the behavior of only a single neuron, because even a single neuron has complex biophysical characteristics and never stops growth. Computational neuroscience is a research field which fills up such a deficiency in experiments. By modeling the essential features of a neuron or a neural network at multiple spatial-temporal scales, we can capture and analyze the fundamental properties of a neuron or a neural network by computer simulation. Moreover, we can even offer some suggestions to experimental study by taking into account the probable results obtained from the simulation.


**RF MEMS**

[29] COMSOL Multiphysics cyclopedia. https://www.comsol.co.in/multiphysics/navier-stokes-

[30] Jain RR, Mehta MR, Bannalikar AR, Menon MD. Alginate microparticles loaded with lipopolysaccharide subunit antigen for mucosal vaccination against *Klebsiella pneumonia*.

equations [Accessed: 02-February 2018]

90 MEMS Sensors - Design and Application

2015;**43**(3):195-201. DOI: 10.1016/j.biologicals.2015.02.001

**Chapter 5**

Provisional chapter

**Study and Design of Reconfigurable Wireless and**

Study and Design of Reconfigurable Wireless and

**Low-Power Applications**

Low-Power Applications

http://dx.doi.org/10.5772/intechopen.74785

Ali Gharsallah

Abstract

Ali Gharsallah

Bassem Jmai, Adnen Rajhi, Paulo Mendes and

Bassem Jmai, Adnen Rajhi, Paulo Mendes and

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

**Radio-Frequency Components Based on RF MEMS for**

DOI: 10.5772/intechopen.74785

This chapter intends to deal with the challenging field of communication systems known as reconfigurable radio-frequency systems. Mainly, it will present and analyze the design of different reconfigurable components based on radio-frequency microelectromechanical systems (RF MEMS) for different applications. This chapter will start with the description of the attractive properties that RF MEMS structures offer, giving flexibility in the RF systems design, and how these properties may be used for the design of reconfigurable RF MEMS-based devices. Then, the chapter will discuss the design, modeling, and simulation of reconfigurable components based on both theoretical modeling and well-known electromagnetic computing tools such as ADS, CST-MWS, and HFSS to evaluate the performance of such devices. Finally, the chapter will deal with the design and performance assessment of RF MEMS-based devices. Non-radiating devices, such as phase shifter and resonators, which are very important components in the hardware RF boards, will be addressed. Also, three types of frequency reconfigurable antennas, for the three different applications (radar, satellite, and wireless communication), will be proposed and evaluated. From this study, based on theoretical design and electromagnetic computing evaluation, it has been shown that RF MEMS-based devices can be an enabling solution in

the design of the multiband reconfigurable radio-frequency devices.

RF resonator, frequency reconfigurable antennas

Keywords: RF MEMS capacitive switch, modeling, tunable, MMIC, phase shifter,

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Radio-Frequency Components Based on RF MEMS for

#### **Study and Design of Reconfigurable Wireless and Radio-Frequency Components Based on RF MEMS for Low-Power Applications** Study and Design of Reconfigurable Wireless and Radio-Frequency Components Based on RF MEMS for Low-Power Applications

DOI: 10.5772/intechopen.74785

Bassem Jmai, Adnen Rajhi, Paulo Mendes and Ali Gharsallah Bassem Jmai, Adnen Rajhi, Paulo Mendes and Ali Gharsallah

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.74785

#### Abstract

This chapter intends to deal with the challenging field of communication systems known as reconfigurable radio-frequency systems. Mainly, it will present and analyze the design of different reconfigurable components based on radio-frequency microelectromechanical systems (RF MEMS) for different applications. This chapter will start with the description of the attractive properties that RF MEMS structures offer, giving flexibility in the RF systems design, and how these properties may be used for the design of reconfigurable RF MEMS-based devices. Then, the chapter will discuss the design, modeling, and simulation of reconfigurable components based on both theoretical modeling and well-known electromagnetic computing tools such as ADS, CST-MWS, and HFSS to evaluate the performance of such devices. Finally, the chapter will deal with the design and performance assessment of RF MEMS-based devices. Non-radiating devices, such as phase shifter and resonators, which are very important components in the hardware RF boards, will be addressed. Also, three types of frequency reconfigurable antennas, for the three different applications (radar, satellite, and wireless communication), will be proposed and evaluated. From this study, based on theoretical design and electromagnetic computing evaluation, it has been shown that RF MEMS-based devices can be an enabling solution in the design of the multiband reconfigurable radio-frequency devices.

Keywords: RF MEMS capacitive switch, modeling, tunable, MMIC, phase shifter, RF resonator, frequency reconfigurable antennas

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.