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Trends in Analytical Chemistry, Vol. 30, No. 6, 2011

Spatially resolved mass spectrometry in the study of art and archaeological objects
Giuseppe Spoto, Giuseppe Grasso
Spatially resolved mass spectrometry (MS) is a widespread technique increasingly used in many areas of research. Operating in a fast, straightforward way with minimal sample damage, MS investigation can provide spatially resolved information with low limits of detection and a wide linear range. For these reasons, spatially resolved MS is applied in the study of art and archaeological objects, and the current review provides a survey of relevant publications. We highlight the main capabilities and the limitations of spatially resolved MS and discuss the most relevant applications. 2011 Elsevier Ltd. All rights reserved.
Keywords: Archaeometry; Atmospheric pressure MALDI; Bronze; Conservation science; Glass; Laser desorption/ionization; MALDI; Mass spectrometry; Pigment; Secondary ion mass spectrometry

1. Introduction
Giuseppe Spoto* Dipartimento di Scienze ` Chimiche, Universita di Catania, Viale Andrea Doria 6, 95125, Catania, Italy Istituto Biostrutture e Bioimmagini, CNR, Viale A. Doria 6, Catania, Italy Giuseppe Grasso Dipartimento di Scienze ` Chimiche, Universita di Catania, Viale Andrea Doria 6, 95125, Catania, Italy

Corresponding author. Present address: Dipartimento di Scienze Chimiche, ` Universita di Catania, Viale Andrea Doria 6, 95125, Catania, Italy. Tel.: +39 095 7385141; fax: +39 095 580131; E-mail: gspoto@unict.it

Analytical chemistry plays an important role in the study of art and archaeological materials, which has a variety of aims. When artifacts are studied, in addition to addressing questions of authenticity, other issues are also investigated (e.g., the technology used to produce them, their distribution from the production areas, and understanding the use to which they were put in the past). By interpreting such information, it is possible to understand better the behavior of ancient people. Long-term storage often tends to obscure chemical information that contributes to the above-mentioned aims. It is thus important to understand, at the deepest possible level, all the processes of alteration that intervene in the life cycle of ancient objects. An important issue to be addressed when valuable pieces of art or archaeological remains are chemically analyzed is how the integrity of the object is affected by the analytical procedure adopted. Analytical techniques are ideally expected to avoid any chemical and physical alteration of the object. For these reasons, analytical techniques able to perform the analysis directly on the object make it unnecessary to take samples and come

close to art curators expectations. In the past, in order to nd a balance between art curators expectations and the requirements of scientic methods, it was necessary: (1) to locate the object in the analyzing chambers of the analytical instrument; or, alternatively, (2) to scrape tiny fragments of samples from its surface. Unfortunately, because art or archaeological objects have to be small to t within the chambers of common analytical instruments, the destructive approach often remains the last resort for the extraction of analytical information from artistic and/or archaeological samples. A large number of new analytical methodologies that better satisfy the requirement for micro-destructiveness or non-destructiveness of the sample to be analyzed has been developed in recent years [1]. In particular, both mass spectrometry (MS) and non-MS spatially resolved analytical techniques provide new opportunities for the micro-destructive study of samples of artistic or archaeological importance [26]. In this context, emphasis should be given to the new possibilities offered by X-ray uorescence (XRF)-based techniques that allow the 3D distribution of different elements in a

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sample to be investigated with spatial resolution in the range 1040 lm [7]. Such methods have been used to image the 3D structure of hidden paint layers. Moreover, the combined use of spatially resolved portable XRF spectroscopy and X-ray absorption has allowed the astonishing sfumato technique used by Leonardo Da Vinci in the Mona Lisa painting to be disclosed [8]. However, spatially resolved MS offers simple sample preparation and a capability for micro-destructive analysis. With minimal sample preparation and damage, MS can be used to investigate biomolecular, organic or inorganic components of works of art or archaeological remains, providing spatially resolved information in a rapid manner. Indeed, the data sets contain information on the distribution of chemical species, which is an advantage in terms of time required compared to methods that require separate tagging or staining for every species of interest [9]. Inorganic MS methods are often more sensitive than XRF in detecting trace components, while Raman microscopy and Fourier transform-infrared (FT-IR) microspectroscopy compete with spatially resolved MS in the study of complex organic matrices when ngerprint spectra of the components are available. The ability of MS to provide information useful for the direct identication of unknown compounds is counterbalanced by the reduced spatial resolution, with which most of the MS methods are able to operate, compared to microRaman and micro-FT-IR spectroscopies. For the reasons outlined above, several studies focusing on the application of different spatially resolved MS methods to the study of works of art and archaeological remains have recently been produced, and we attempt to organize and to discuss the results achieved so far.

penetration of the incident radiation, dened as the inverse of the absorption coefcient, has to be considered. Lateral resolution is similarly dened for both surfacesensitive and non-surface-sensitive analytical techniques and refers to their ability to provide independent signals from different areas of the solid sample. The minimum distance of two areas on the sample surface generating a signal level of 16% and 84% or, under more stringent conditions, a signal ratio of 104, is used to quantify the lateral resolution of MS analytical techniques [12]. Spatially resolved analytical techniques mostly operate with a typical lateral resolution in the range of microns [2]. The novel, useful aspect of spatial resolution is to image the distribution of elements or compounds across a sample surface. The direct analysis of microscopic areas of a work of art may be accomplished by lateral resolution, thereby allowing the extraction of a wide range of valuable analytical information that can be correlated with an image. A small lateral resolution also makes possible the analysis of tiny fragments of samples scraped from the object of interest with minimal damage to the artifact itself. The ability to resolve analytical data spatially is mostly used to collect information from a specic region of the sample within an area usually in the range tenths to hundreds of lm2. In this case, localized analysis with a lateral resolution that is a direct consequence of the ability to focus the incident and/or the emitted beam of lighter particles is obtained.

3. Role of MS in studying works of art and archaeological remains Analytical techniques used for the study of materials or objects of artistic or archaeological importance are expected to provide a wealth of information on: (1) the chemical composition or nature of the material under study; (2) the state of alteration of the objects caused by the long-term exposure to the environment; and, (3) the effects of restoration procedures. All the above information must be extracted from samples that are a complex mixture of different chemical compounds. Ancient artifacts were manufactured by manipulating natural products that were often a mixture of both organic and inorganic compounds. The processes of change that intervene in the life cycle of the artifacts contribute to increasing further their chemical complexity. In this scenario, the great potential of MS to identify chemical species present in complex mixtures has been widely used in the study of works of art and archaeological remains. Inorganic MS has been particularly useful for chemical ngerprinting, as provenance of archaeological materials is often inferred by evaluating results from trace-element analysis or from quantitative determination of the relative
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2. Spatially resolved methods The spatial resolution of an analytical technique denes the ability to extract and to separate analytical information from different regions of a sample. Most of the spatially resolved analytical techniques applied in the study of works of art and archaeological remains involve probing the sample by exposing it to a ux of particles (photons, electrons, ions, neutrals) and simultaneously measuring the energy distribution of a specic class of particles emitted or scattered [10,11]. Each spatially resolved analytical technique operates with a specic lateral resolution. Moreover, depth resolution is also used in order to characterize the vertical performance of surface-sensitive analytical techniques and it is dened by the distance between the 84% and 16% levels of the depth prole the 2r limits of the Gaussian distribution of the detected signal at the layers interface of a selected element or compound sandwiched between two differently composed layers [12]. In the case of laser-based techniques, the depth of

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amounts of selected isotopes [13]. Inorganic MS has also been applied to identify pigments in paints or the main constituents of glass and ceramic objects, enamels and glazes [14]. However, organic MS has been widely used to deal with a variety of different problems concerning the identication of organic and biomolecular compounds constituting artistic, historic and archaeological objects [15]. The organic raw materials used in both traditional art and archaeological contexts are, for the most part, natural products, and, hence, composed of complex mixtures of inorganic, organic and biomolecular components [16]. Moreover, the intentional mixing of organic binders and pigments during the preparation of painting materials or repeated use of archaeological cooking vessels to prepare a range of different foodstuffs are two examples of human activities that introduce further complexity into the chemical composition of the raw materials. Further increases in the complexity of composition derive from the transformation of the original natural materials generated by the degradation processes occurring through material use and environmental exposure [e.g., proteic materials used in traditional tempera painting are subject to condensation and cross-linking reactions with other components (e.g., lipids)]. The unparalleled capacity of MS to reveal information useful for identifying the organic components of artistic, historic and archaeological objects with further possibilities for their detection and quantication at trace levels in complex matrices derives from the ability to couple chromatographic techniques to mass spectrometers, thereby allowing separation of the individual molecular species with on-line MS analysis of the eluting components [17]. A similar approach was adopted to quantify the compound-specic carbon (d13C) and hydrogen (dD) isotoperatio values from fatty-acid residues discovered on potsherds from Kazakhstan and attributed to the Eneolithic Botai Culture (about 3500 BCE) [18]. The study provided the earliest evidence of horse domestication in antiquity. In some cases, an interdisciplinary approach is necessary if different components of precious samples have to be identied. For example, in order to assess the components of ancient cosmetics and to obtain better understanding of their preparation, complementary MS techniques were exploited (e.g., pyrolysis GC-MS and other analytical approaches) [19]. It was therefore possible to identify different preparation procedures of two Roman esh-colored make-ups that were preserved on the surface of a Roman bronze cosmetic tool and in a glass unguentarium. Organic MS is also widely applied for restoration or conservation purposes. When studying paint layers in artworks, the investigation of their complex organicchemical composition aims to identify both original constituent materials and those components formed as a consequence of aging. Such study is of great importance to select the most appropriate conservative strategy. 858
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In this context, GC-MS has been shown to identify lipids, waxes, proteins, and resinous materials used in creating painted works. In particular, the potential offered by GC-MS when combined with proper chemical procedures was demonstrated using 0.20.9-mg samples scraped from: (1) the painted hull of a shipwreck dated to the late 1700s/early 1800s; (2) an Italian painting on canvas produced between late 1600s and early 1700s; and, (3) a painted Etrurian sarcophagus [20]. Changes in the chemical composition of paintings can also occur in museum environments. Tempera paints suffer from alterations mostly involving the varnish layer and the binding medium. Cholesterol-oxidation products (COPs) have been shown to result from light-ageing of egg-binding media and their presence was proposed as a marker of light-induced degradation of the binding medium [21]. Direct-temperature-resolved MS (DTMS) analyses of samples taken from painted German baroque altars and from the Red on Maroon mural painting stored at Tate Modern London have conrmed the presence of COPs.

4. Spatially resolved MS In the examples discussed above, the MS-based analyses required fragments of solid samples (typically 110 mg) to be taken from the object to be investigated. However, in recent years, MS techniques that use spatially resolved ionization sources have been applied for the microdestructive study of artistic and archaeological objects. Secondary ion (SI) MS uses ion beams in order to generate charged species from the sample surface while laser desorption ionization (LDI) and laser ablation (LA) methods rely on the use of photons generated by laser sources. The lateral resolution in these methods is improved by focusing the incoming primary beam. Table 1 shows the typical lateral resolution and depth of penetration for the spatially resolved techniques used in the study of art and archaeological objects discussed in this review with other information related to the destructivity of the method and the XYZ translation limit. The latter information is important in order to evaluate the volume of the 3D objects that can be analyzed by using each of the discussed spatially resolved technique.

4.1. Spatially resolved SIMS Ion sputtering is the physical process underlying SIMS detection. It relies on a cascade of atomic collisions generated when energetic ions hit the surface of the solid target. The energy deposited by the bombarding ions is partially reected back to the surface through target

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atoms mobilized by the impact of incident ions or through cascade collisions. When the energy of the recoiled atoms near the surface is greater than the binding energy of the surface atoms, these are ejected into the vacuum environment. The interaction volume of the primary incident ions with the solid surface depends upon the energy of the primary ion beam and the target composition, but the sputtered species originate from no more than the rst two atomic layers. For this reason, SIMS is a very surface-sensitive analytical technique. SIMS, in principle, is capable of chemically analyzing and imaging both inorganic and organic components that are present at major, minor or trace levels in solid samples with a lateral resolution as small as 50 nm [22]. Scanning electron microscopy/energy dispersive X-ray uorescence spectroscopy (SEM-EDXRF) [23], particleinduced X-ray emission (PIXE) [24] and laser-induced breakdown spectroscopy (LIBS) [25] rival SIMS in their spatial resolution of inorganic components of works of art and archaeological objects. However, SIMS has signicantly lower limits of detection (LODs) (in the range 0.011 109 atoms/cm2) and a much lower depth penetration. SIMS also has smaller lateral resolution compared to microLIBS (315 lm) [26] and SIMS allows chemical images of the analyzed surface to be obtained using specic ion-detection modes. A distinction is made between the static (s) and dynamic (d) SIMS operational regimes, each of which yields different information and analytical features. In order to understand the basic differences between the two regimes, we should consider that a primary ion ux of 1015 ions/cm2/s is sufcient to sputter more than one atomic layer/s from the target surface. If the primary beam is typically focused over an area of the target surface of about 104105 lm2 and the acquisition time is longer than 1 s, then an analysis carried out by using a continuous primary beam provides information on a surface renewed continuously by the sputtering process. Similar operating conditions dene the dSIMS regime that is useful for studying the depth-prole composition of the sample [27,28]. When the primary ion ux is decreased to 10111013 ions/cm2/s, so that only secondary ions sputtered from a fraction of an atomic layer are collected, the static regime is established. Under this regime, sSIMS operates as a surface-specic analytical technique, since only information from the outer atomic layer of the target is obtained. Major drawbacks for SIMS analyses of ancient objects are the required ultra-high vacuum environment (109 1011 torr) and difculties with quantication procedures. Moreover, the instrumentation is not mobile. For LIBS [25,29], compact equipment can be used and the analysis can be conducted on site (e.g., the museum, the conservation laboratory, or even outdoors at an excavation site). Nevertheless, sample requirements for SIMS analysis are not particularly stringent. Although sample

size is a signicant limitation, solid samples are usually directly analyzed with no special preliminary treatments. Samples of about 2030 mm in diameter and no more than 5 mm in height are allowed with traditional SIMS equipment, although the sample must tolerate the SIMS ultra-high vacuum environment. Such a requirement is particularly critical, since desorption of gaseous components in the ultra-high vacuum chamber can affect the integrity or the shape of organic objects (e.g., manuscript fragments or painting samples). A nal remark concerns sample conductivity, as conducting samples are analyzed more easily. Nevertheless, surface charging that occurs when non-conducting samples are analyzed can sometimes be prevented by using very low primary-ion doses or electron-ooding procedures. The surface sensitivity of sSIMS can be a severe problem when dealing with weathered or corroded materials. Change processes modify the surface composition to such a degree that the information obtained may be considered representative of only the top few monolayers of the altered surface. However, studies aimed at identifying weathering processes in their early stages may greatly benet from the use of this surface technique. Surface-sensitivity limitations may also be partly overcome by using the depth proles that dSIMS can provide. SIMS was rst used in the study of art and archaeological objects in the 1980s, and a number of applications have revealed its potential and limitations in the study of ancient materials [30]. A detailed description of such studies may be found in review articles published in the past few years [3032]. The wide elemental range that can be investigated (potentially from H to U), its excellent LODs and the quantitative information obtainable when using SIMS are just some of the advantages of this technique. The use of time-of-ight (ToF) mass analyzers has improved the performance of SIMS in terms of analyzed mass range, surface sensitivity and mass resolution. Coupling ToF mass analyzers improved performances, as the static conditions allowed by the pulsed ion source used in ToF-SIMS has expanded the application of SIMS to the study of a variety of organic, polymeric and biological materials. Metals and alloys are, in principle, good materials for SIMS analysis. They are electrically conductive, can easily be handled so as to obtain clean, at and smooth surfaces for analysis, and are also usually compatible with ultra-high vacuums. However, archaeological and/ or artistic metal objects do not always possess all of these properties due to degradation processes, which may chemically modify both the surface and the interior of samples. Non-conductive patinas and inltrated compounds may often form as a result of long burial in soil or exposure to the atmosphere, and these might have very different characteristics compared to those observed for pure or alloyed metals.
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Table 1. Summary of some relevant characteristics of the spatially resolved methods discussed LA-ICP Typical lateral resolution Typical depth of penetration 5300 lm [47] $100 nm per laser pulse [47] XY = 5 cm; Z = 2 cm 760 torr Argon gas 50500 lg micro LIBS 315 lm [26] 100500 nm [29] LDI-UV 200500 lm 50200 nma [51] MALDI-IR 100300 lm [51,52] 1.52.0 lm [51] MALDI-UV 50200 lm [51] 50200 nm [51] AP-MALDI $400 lm [71] 50200 nm [51] dSIMS 50100 nm [27] 25 nm [28] sSIMS 1050 nm [21] 0.050.5 nm

Typical XYZ movable rangeb Operating pressure Removed material

XYZ = 20 cm 760 torr air 0.010.2 lg [25]

XY = 20cm Z=0 107108 torr A few tens of lg of material is sampled from the object to be analyzed [45] Organic and inorganic pigments and dyes.

XY = 20cm Z=0 107108 torr Fmol per pulse laser [50]

XY = 20cm Z=0 107108 torr Amol per pulse laser [51]

XZ = 50 cm; Y = 30 cmc 760 torr air 0.33 nmoles/mm2 [71]

XY = 1030 cm; Z = 25 cm 1091011 torr $pg

XY = 1030 cm; Z = 25 cm 1091011 torr A few atomic or molecular monolayers

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Applications of analysis

Trace-element and isotope of metals, pottery and minerals

Elements of metals, pottery, glass, stone, soil, pigments

Organic components and residues

Organic components and residues

Organic components: pigments, dyes, binders, resins

Glass degradation, ancient coin plating, metallic layers, obsidian hydration dating

Surface of pigments, pottery, metals, alloys, and minerals

b c

Most of the LDI-MS experiments were carried out using standard MALDI equipment. For this reason, lateral and depth resolution similar to MALDI were obtained. Improved performances in term of lateral resolution were obtained using specically designed laser-microprobe equipment [51]. The values shown refer to standard commercial equipment. The reported values refer to equipment specically designed for the analysis of art objects [71].

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Several metal objects of archaeological and artistic interest have been studied using SIMS. These include bronze artifacts [33,34], ancient silver coins [35], and antique iron music wires [36]. SIMS has been frequently used in the study of the deterioration of glass objects [37]. Glass is commonly susceptible to a variety of reactions that affect its durability. Ancient glasses most clearly show the consequences of their relatively limited resistance to attack by water, acid and alkaline aqueous solutions, steam, or atmospheric agents. Long exposure to change agents worsens the consequences of degradation, which is also seen to affect modern glasses. Glass weathering specically involves attack by water combined with environmental and biological agents. Water corrosion is sparked off by an ion-exchange mechanism involving the alkali ions in the glass and hydrogen from the environment. SIMS has been used to study a variety of ancient glasses with the aims of understanding the degradation process [38], provenance [37] and dating [39]. The use of both spectroscopic and imaging SIMS in the study of pigments and binding media used in paintings have shown the potential of SIMS to characterize and to image both inorganic and organic species [40,41]. SIMS has been used to image pigment distributions in cross sections of paint, thus contributing to the identication of the different techniques used in traditional painting [42,43]. In another study, it was possible to examine variations in the composition of the top and the bottom of a paint lm, and the spatial correlation between metal and fatty-acid composition in metal-soap aggregates [40]. 4.2. Spatially resolved LDI-based MS Spatially resolved MS analysis of artistic or archaeological objects can also be carried out by desorbing charged species from the sample surface with a laser beam. The switch from primary ions to laser beams signicantly reduces the lateral resolution, typically going from the sub-micron range to the tens-of-microns range (Table 1). However, it is possible to control the laser-beam diameter and focus in order to improve the spatial resolution of the analysis. The use of far-UV or IR laser sources operating with pulse durations in the ns range has been shown to be important for the direct laser desorption of charged organic species with molecular weights of about 12 kDa [44]. Moreover, the loss of spatial resolution occurring when laser beams are used instead of primary ions is counterbalanced by the less demanding vacuum requirements that translate into enhanced possibilities for the direct analysis of works of art and archaeological objects. For this reason, LDI/MS is often applied as a complementary analytical tool for use in the conservation laboratory alongside other techniques {e.g., Fourier transform infrared (FTIR) spectroscopy and Raman microscopy [45]}. An advantage of LDI/MS over Raman microscopy is that, in many cases, by using both positive

and negative ion modes, LDI/MS provides sensitive, specic identication of organic systems based simply on exact masses of fragment ions and isotopic patterns. The laser-desorption regime is established when a solid sample is irradiated with laser uencies of 0.011 J/cm2. Under such conditions, sample vaporization and partial ionization begins. Greater laser uence causes pronounced decomposition of the irradiated surface. In addition, the development of large stress waves due to the thermally-induced expansion can result in solidmatrix disintegration and the collective ejection of large molecular clusters [46]. This phenomenon, known as laser ablation, is exploited by laser ablation-inductively coupled plasma (LA-ICP)-MS in order to perform multielemental and isotopic analyses of solid samples. LA-ICPMS is very suitable for studying art and archaeological objects because it offers the exibility to achieve spatially resolved information with low LODs and a wide linear range with minimal sample damage. This technique is mainly used to obtain multi-element information at various concentration levels, giving elemental or isotopic ngerprints that could help to answer relevant archaeological questions, often concerning samples such as coins and pottery. However, as this topic has been very recently reviewed [47], we do not discuss it further. Nowadays, the possibilities offered by direct LDI-MS in the study of non-volatile compounds maintained under vacuum are well known [48], and limitations in investigating large molecules have been established. However, some limitations suffered by LDI-MS were circumvented in 1987 with matrix-assisted (MA)LDI-MS [49]. In MALDI, the analyte compounds are embedded in a surplus of an organic compound (matrix) that is co-desorbed upon laser excitation. This compound is chosen according to the analyte properties and the laser energy [50]. The matrix provides a number of different functions, including: (1) isolating the analyte molecules and preventing their aggregation; (2) absorption of the laser energy via electronic or vibrational excitation (depending on the laser energy); (3) evaporation of unchanged analyte molecules; and, (4) efcient ionization of analyte molecules [51,52]. MALDI sources are usually coupled with ToF mass analyzers because of the inherently pulsed nature of the short temporal (ns) laser sources. The small spatial distribution of desorbed ions further favors the use of ToF analyzers, giving high mass resolution (M/DM % 10,000), high mass accuracy (a few ppm), and low LODs [53]. However, recently, similar performances have been also obtained by using ion-trap mass analyzers [54], providing an easier access to MS2 analyses. 4.3. MALDI and atmospheric pressure MALDI LDI and MALDI experiments are typically conducted at the low pressures found in most ion sources (107 Torr).
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This environment is useful for ion transfer to the mass analyzer and inuences the mechanism by which ions are formed. However, MALDI spectra are also obtained at higher pressures, including atmospheric pressure (AP). A preliminary report published in 2000 [55] showed that AP/MALDI produced ion currents comparable to that in electrospray (ESI) by using a nitrogen laser operating with a 20-Hz frequency and an energy density of 2675 mJ/cm2 per pulse. More recently, AP/ MALDI experiments using IR sources have also been performed [56]. In the past several years, AP/MALDI has been implemented with a number of mass analyzers, including ToF [55], ion trap [57], and Fourier-transform ion cyclotron resonance (FT-ICR) [58], taking advantage of placing the AP/MALDI ion source externally to the mass analyzer. Such a conguration is particularly suitable for the analysis of artistic and archeological material, as it makes analysis of 3D objects easier [59]. AP/MALDI analyses are carried out in a manner similar to that for conventional MALDI, with a solid matrix co-crystallized with the analyte. This requires the sample to be destroyed or modied. Nevertheless, AP/ MALDI spectra can, in some instances, also be obtained simply after depositing a matrix layer on the sample surface. Ions generated by AP/MALDI have been shown to be less energetic than those produced by MALDI and are thus subject to signicantly less metastable decay due to fast thermalization of the internal energy of the ion at atmospheric conditions. However, the reduced ion transmission from the atmosphere into the low vacuum of the mass analyzer gives lower ion currents compared to conventional MALDI. Ion-transmission efciency depends on both laser-spot displacement relative to the central axis of the mass-analyzer inlet capillary and the voltage applied between the target plate and the inlet capillary [60]. A signicant increase in ion transmission is obtained by adopting an approach called pulsed dynamic focusing (PDF) that involves switching the electric eld to zero for a time interval during which ions approach the capillary inlet [61]. Spatially resolved MS techniques based on direct LDI have been employed in the study of art and archaeological objects since 2002 [62,63]. However, as discussed in previous sections, direct LDI is only effective in the study of a limited range of materials that can be volatilized, while MALDI has expanded LDI-based MS methods to studies of organic components from works of art. Examples of the applicability of MALDI to the analysis of pigments [64], siccative oils [65], proteinaceous binders [6668], and varnishes [69] have been reported in the literature. A proteomic approach based on the use of MALDI-ToF was also used to identify biological samples that are more than 5300 years old, taken from the Tyrolean mummy, also called Iceman or Oetzi [70]. 862
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Recently, possibilities offered by AP/MALDI for analyzing artistic material directly in air have been demonstrated by studying traditional painting materials, iron-gall ink [71] and organic dyes and pigments used to print books dated 191120 [59]. This last study denitely demonstrated the applicability of AP/MALDI to the study of ancient objects, because the analyses were carried out in situ with a spatial resolution of about 400 lm. This was achievable by placing books in a modied AP/MALDI source on an XYZ translation table. The microdestructivity of the analysis was obtained by spraying about 0.2 lg/mm2 of the matrix (corresponding to about 0.8 nmole/mm2) on a 23-mm2 area of the sample.

5. Conclusions Spatially resolved MS analytical techniques have great potential for the study of art and archaeological objects. In particular, the techniques described here have several important characteristics: (1) capabilities for spatially resolved multi-elemental and isotopic analysis; (2) low LODs and extended linear ranges that allow quantitative analysis; (3) minimal sample preparation and/or damage; and, (4) capabilities for spatially resolved analysis of organic and biomolecular compounds. We have given special mention to recent instrumental advances that allow direct, spatially resolved MS analyses of works of art. Such facilities open new possibilities for complex artistic and archaeological objects when direct MS analyses of the studied object are conducted at AP.

Acknowledgments We acknowledge support from MIUR (FIRB RBRN07BMCT). We would like to thank D. Anglos, D. Garozzo and an anonymous referee for their constructive comments and suggestions.

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