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Synchrotron UV-Visible Multispectral Luminescence Microimaging of Historical Samples Mathieu Thoury,†,4 Jean-Philippe Echard,‡ Matthieu Refregiers,§ Barbara Berrie,† Austin Nevin,^,z Frederic Jamme,§ and Loïc Bertrand#,* †
Scientific Research Department, National Gallery of Art, Fourth and Constitution Avenue NW, Washington D.C. 20565, United States Laboratoire de Recherche et de Restauration, Musee de la Musique, Cite de la musique, 221 avenue Jean Jaures, F-75019 Paris, France § Synchrotron SOLEIL, DISCO Beamline, BP48 Saint-Aubin F-91192 Gif-sur-Yvette cedex, France ^ Dipartimento di Fisica, Politecnico di Milano, Piazza Leonardo da Vinci 32, Milano, 20133, Italy z IFN-CNR, Piazza di Leonardo da Vinci 32, Milano, 20133, Italy # IPANEMA UPS 3352 CNRS, Synchrotron SOLEIL, BP48 Saint-Aubin, F-91192 Gif-sur-Yvette cedex, France 4 Centre de Recherche sur la Conservation des Collections, Museum National d’Histoire Naturelle, UMR 7188 CNRS, CP 21 36 rue Geoffroy Saint-Hilaire, F-75005 Paris, France
Anal. Chem. 2011.83:1737-1745. Downloaded from pubs.acs.org by KAROLINSKA INST on 01/23/19. For personal use only.
‡
ABSTRACT: UV-visible luminescence techniques are frequently used for the study of cultural heritage materials, despite their limitations for identification and discrimination in the case of complex heterogeneous materials. In contrast to tabletop setups, two methods based on the vacuum ultraviolet (VUV)UV-visible emission generated at a bending magnet of a synchrotron source are described. The main advantages of the source are the extended wavelength range attained, the continuous tunability of the source, and its brightness, leading to a submicrometer lateral resolution. Raster-scanning microspectroscopy and full-field microimaging were implemented and tested at the DISCO beamline (synchrotron SOLEIL, France). Investigative measurements were performed on a sample from a varnished musical instrument and a paint sample containing the pigment zinc white (ZnO) in order to illustrate some of the challenges analyzing heterogeneous cultural heritage cross-section samples with the novel imaging approach. The data sets obtained proved useful for mapping organic materials at the submicrometer scale and visualizing heterogeneities of the semiconductor pigment material. We propose and discuss the combined use of raster-scanning microspectroscopy and full-field microimaging in an integrated analytical methodology. Synchrotron UV luminescence appears as a novel tool for identification of craftsmen’s and artists’ materials and techniques and to assess the condition of artifacts, from the precise identification and localization of luminescent materials.
T
he chemical characterization of materials from artifacts of cultural heritage significance presents a variety of challenges for conservation scientists. Cultural heritage samples are often intrinsically heterogeneous at the microscale due to the diversity of original compounds and their complex morphology. In crosssections taken from heritage artifacts, such as paintings or musical instruments, stratified layers of 1-25 μm thick are composed of mixtures of organic and/or inorganic materials that may have undergone chemical modifications and aging. Alteration features of crucial relevance may be limited to the boundaries between originally distinct materials. In this context, the use of analytical probes providing unambiguous signature and spatial distribution of materials at a submicrometric length scale is required to correlate with the characteristic heterogeneity length scales. A balance must be found between the high spatial resolution required to work at a scale where the complexity of the mixtures is low enough to understand the sample features and the extreme sensitivity required to analyze low quantities of matter typically ranging from pico- to femtograms in (μm)3 volumes. A full understanding of the methodologies and the chemical condition of materials used in r 2011 American Chemical Society
archaeology and cultural heritage requires study ranging from the macroscopic to the molecular level via microscopic and mesoscopic analyses of such small volumes. Current and promising developments in the study of the composition and spatial organization of organic binding media in complex microsamples include conventional FT-IR imaging,1-5 ToF-SIMS,6 nanoscale FT-IR near-field imaging,7 multiplexed chemiluminescence immunolocalization,8 immunofluorescence detection8,9 and synchrotron scanning transmission X-ray microscopy.10,11 We previously employed nondestructive techniques including FT-IR microspectroscopy12-14 and imaging14 to localize at the micrometer-scale different classes of organic binding media in cross-sections from historical musical instruments. We have also investigated the potentials of Raman spectroscopy15 and luminescence spectroscopy16-19 to discriminate between organic binding materials on model samples at the Received: November 12, 2010 Accepted: December 26, 2010 Published: February 03, 2011 1737
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Analytical Chemistry macro- and mesoscales. At the microscale, it appears that we lack nondestructive methods for the discrimination among different classes of organic materials, such as between proteinbased materials. Methods such as micro-Raman spectroscopy, micro-X-ray diffraction (μXRD), and micro-X-ray absorption near edge spectroscopy (μXANES) are increasingly being used on cross-sections for specific identification of materials and for the observation of chemical characteristics that may provide information relative to manufacturing and aging processes.20-22 However, methods are still sought that provide complementary physicochemical information on inorganic heritage materials in heterogeneous cross-sections containing pigments in sometimes low concentrations, with submicrometer particle size, or in very thin layers. In particular, the identification of structural defects and the characterization of the distribution of these defects might provide a powerful way to better understand historical sources of raw materials, manufacturing treatments, and processes. UV-visible-induced luminescence of materials was one of the earliest tools available to enhance the contrast and therefore discriminate between supposedly original and restoration materials on the surface of objects, which otherwise might be barely perceptible under visible light, but only recently has luminescence spectroscopy been proposed for chemical analysis of cultural heritage. As early as 1931, pioneering work was reported on the use of a UV lamp to examine polychrome objects in situ, in organic binding media on panel paintings, and on ancient stones.23-26 The growing availability of UV lamps allowed the application of the technique in conservation studios, by professional dealers, in auction houses, and by museum curators and conservators to examine paintings, gems, furniture, or musical instruments.27 The intensity and the color of luminescence in the visible could be observed and photographically recorded for consultation and archival purposes.28 A number of artists’ materials were found to emit in the infrared range using photographic films sensitive beyond the visible range.29 It is only from the beginning of the 1980s that luminescence spectroscopy, using a variety of light sources (medium-pressure Hg lamps, He-Ne and Arþ lasers), was applied to the study of artists’ materials, such as resins, oils, and lake and inorganic pigments.16,17,30-38 These works have shown that the luminescence properties from these materials are often complex and may change with aging and upon mixing, which complicates the use of luminescence spectroscopy for the positive identification of artists’ materials. The development of luminescence imaging systems was a corollary of the availability of sensitive multichannel photodetectors which can be used to record the spatial distribution of luminescent materials.39,40 Fluorescence lifetime imaging (FLIM), using gated photodetectors, added the temporal component to the measurement to help separate more finely luminescence signals from different chemical moieties, thereby refining the chemical characterization of materials in wall paintings.41,42 Recently, a calibrated luminescence imaging spectroscopy setup was developed by Delaney et al. and was used for the identification and mapping of the cadmium yellow pigment in a painting by Pablo Picasso.43 Along with the use of luminescence to detect heterogeneity and diversity of materials in situ on cultural artifacts, luminescence microscopy has mainly been used as a visual, qualitative technique to help reveal the structures of complex layering or mixtures in micrometric samples, exploiting the differences in the color of emissions.44 The excitation range available for microscopic analysis was later expanded to the visible region with the
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use of dichroic filter cubes, which allowed the visualization and localization of certain organic binders and colorants.45 Luminescence microspectroscopy has been applied to the study of binding media and varnishes in cross-sections46 and, more recently, to the study of organic dyes also in cross-sections. The collection of luminescence spectra allowed the identification of lake pigments and dyes.47,48 Achievement of high spatial resolution measurements showing good signal-to-noise with luminescence microspectroscopy is dependent on several parameters, related to the nature of the materials studied and to the specifications of the instrument used. Several factors influence the analysis spot size in luminescence microspectroscopy. Instrumentation-related factors include (a) the irradiance at the sample, at a given excitation wavelength and excitation bandpass, (b) the interaction volume linked to the objective numerical aperture, and (c) the transmission properties of the optical components and the detector sensitivity over a given emission spectral range; interdependent factors depending from the intrinsic nature of the sample include (d) the luminescence yields and concentration of the luminophores under study and (e) optical effects from the matrix containing the luminophores. Spectra with good signal-to-noise ratios were obtained from paints containing lake pigments with 8 μm diameter spots on model samples and 30 μm diameter spots on historical samples.49 In some cases, adequate spectra could be obtained on spots as small as 4 450 or 5 5 μm2.51 In this work, we present an approach based on a VUV-UVvisible multispectral luminescence microimaging setup using a synchrotron source, applied to historical materials in microsamples. We will use the term luminescence, gathering both the fluorescence and phosphorescence processes. In the literature, similar techniques have been described using a variety of terms including microspectroscopy, microspectrofluorimetry, and fluorescence microscopy. We will make the distinction here between techniques, according to the way data were collected: thus, luminescence microspectroscopy will relate to the setup providing spectra from spot analyses and maps generated by raster-scanning, whereas multispectral full-field luminescence microscopy will relate to 2D images collected in full-field mode in multiple spectral ranges, both techniques being jointly designated as multispectral luminescence microimaging. We present two examples of the advances made toward complete characterization of materials in samples from historical works. In these examples, prior work gave valuable information, which, however, was significantly limited by analytical constraints. In the case of the pigment zinc white, information was limited to the identification of the compound from the band edge excitonic emission. Here we show how synchrotron VUV-visible luminescence microspectroscopy provides information on the presence of particles within the sample that have specific crystal defects or impurities, which was previously unobtainable for paint samples. In the second case, the use of a proteinaceous layer in the varnish system on a historical musical instrument had been established; however, information on the type and specific distribution of protein materials within the coating was limited.14 Using luminescence microspectroscopy, we characterized the protein-based stratum. We show that the new synchrotron-based setup allows a 10fold improvement in lateral resolution and, usefully, narrow excitation wavelength ranges down to fractions of a nanometer, while maintaining a sufficiently high beam intensity to attain highresolution mapping of luminescence in small samples. We will discuss the current and future capabilities in the development of 1738
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Figure 1. Synchrotron VUV-UV-visible techniques presented in this work. Two approaches were used, using different microscope setups: (A) luminescence microspectroscopy. Note the adjustable pinhole before the emission detection system. (B) Multispectral full-field luminescence microscopy. Regions of interest in the emission spectrum are selected using an emission filter wheel.
combined luminescence microspectroscopy raster-scanning and multispectral full-field luminescence microscopy.
’ METHODOLOGICAL RESULTS Developments based on synchrotron VUV-UV-visible microimaging were implemented and tested at the DISCO beamline of the SOLEIL synchrotron.52 The white photon beam of the 1.7 T bending-magnet at the beamline is collected and monochromatized by an iHR320 monochromator (Horiba) equipped with a 100 grooves/mm grating blazed at 250 nm, and 0.3 nm fixed entrance and exit slits. The monochromatic excitation beam energy could be continuously tuned in the 180 (VUV) - 600 nm (visible) wavelength range. The resulting monochromatic beam was injected into two inverted UV apochromatic microscopes (Figure 1).53 For both setups, samples (thin sections and paint samples of 1 μm and 1 mm thickness respectively) were deposited onto quartz coverslips. Coherence in light transmission between the objective and the quartz coverslip was ensured by a drop of glycerin that does not contact the sample. Synchrotron Luminescence Microspectroscopy. Synchrotron luminescence microspectroscopy was performed using an inverted IX71 microscope (Olympus) with a custom-made lens set transparent in the deep UV (Figure 1A). Two glycerin immersion objectives were used: a 100 Ultrafluar (Zeiss; numerical aperture (NA) = 1.25) for high magnification and resolution and a 40 Ultrafluar (Zeiss, NA = 0.6) for a larger probing voxel; both objectives were corrected for the 170 μm thick quartz coverslips. A dichroic mirror with an average of 50-50% reflection and transmission over a broad energy band was used. An adjustable pinhole, defining the area from which luminescence was collected, was placed ahead of a triple monochromator. The
layout of the transfer optics to the microscopes and from the microscopes to the detectors is identical to that described by F. Jamme et al.53 The full spectrum is dispersed on 1024 pixels onto a Peltier-cooled iDus CCD (1024 256 pixels, Andor) with a 26μm pixel size. The two-dimensional rastering is ensured by a P542.2 CD nanopositioning XY stage (PI). Data were processed using the software LabSpec (Horiba Scientific). Semiconductor pigments, such as cadmium or zinc oxide pigments, were widely used by 19th and 20th century artists, including Vincent Van Gogh and Pablo Picasso. The case study reported here is the first step toward the setting up of an analytical procedure for characterizing semiconductor pigments (such as ZnO, ZnS, CdS1-xSex, TiO2, etc.) in historical painting cross-sections based on their luminescence properties. A naturally aged paint sample prepared in 1976, made of ZnO pigment (Sennelier France, Paris) dispersed in linseed oil, was analyzed. The emission spectrum is composed of three main bands with maxima at 383, 410, and 510 nm (Figure 2A). The emission band at the shorter wavelength (383 nm) is due to near band edge excitonic recombination54 while the broad weak emission in the 500-600 nm region, which has been attributed to oxygen vacancies in the crystal, is known as the ‘trap state’ emission.55 A 260 nm excitation wavelength predominantly induces luminescence from the zinc oxide and minimizes that from the binder, which is very weakly luminescent at this excitation wavelength.18 It cannot be excluded that the binder slightly contributes to the weak emission observed between 450 and 580 nm (Figure 2A). Photoluminescence properties of ZnO are strongly correlated to its crystal structure and orientation.56 The ratio of intensities of the emissions (band edge/trap state) has been reported to be indicative of the number of defects in the crystals.57 Over distances of fractions of a micrometer, the intensity of the band edge emission 1739
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Figure 2. (A) Synchrotron luminescence microspectroscopy spectrum of ZnO paint film from a spot measurement; 280 nm excitation, 10 s integration time. The adjustable pinhole aperture was 300 μm. (B, C) Maps produced in raster-scanning mode (50 pixels 50 pixels) over 10 10 μm2 of the paint film by integrating the signal intensity without baseline subtraction in the following ranges: (B) 370-405 nm and (C) 400-440 nm. Maps were oversampled with 200 nm steps in X and Y and a beam size of ca. 800 nm; 280 nm excitation, 10 s integration time. Each map was normalized from its minimum to its maximum intensity value, respectively, set to 0 (black) and 1 (white).
varies by 3-fold or more, the ZnO distribution being uneven (Figure 2B). No intensity variations (monitored using the Rayleigh line intensity at 280 nm), which would have indicated significant sample roughness, were detected. Particles between 1 and 3 μm in length are associated with an additional intense luminescence emission between 400 and 430 nm with a maximum centered at around 410 nm (Figure 2C). Notably, at this particle size, this signal does not correlate to a decrease in the intensity of the band edge emission that may have signaled the presence of a distinct chemical compound. This emission feature may therefore be indicative of a distinct crystal defect within ZnO crystals, rather than the presence of a different compound. Indeed, similar emission bands in the 400 to 420 nm range have been observed at the macro-scale on pure synthesized ZnO nanocrystals.58 We show here that synchrotron luminescence microspectroscopy allows resolving spatially, at the submicrometer scale, the luminescence of pigment grains embedded in a paint binding medium, and acquiring a deeper understanding in the characterization and the distribution of crystal defects among the pigment particles. The potential of synchrotron luminescence microspectroscopy in the more complex case of the identification of organicbased media in cross-section samples is illustrated here with the analysis of a sample of varnish from a cello made by Jacques Boquay, a prominent Parisian instrument-maker, at the beginning of the 18th century, who was a contemporary of the famous violin-maker Antonio Stradivari. This varnish system, made of two strata applied
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onto wood, had been previously extensively investigated by FT-IR microspectroscopy, FT-IR full-field microscopy, and gas chromatography.14 While FT-IR data indicated the proteinaceous nature of the underlying stratum, these data alone did not allow more precise characterization of the specific nature of the protein-based material used, i.e. whether the materials are derived from albumin, or collagen derivatives (from cartilage, bones, and skin). Amino acids analysis had then to be performed by GC/MS to provide the identification of a gelatin-based material, probably ‘animal glue’. However, since destruction of the sample is required for this type of separative analysis, information on the relative distribution of materials throughout the layered structure is lost. Using the synchrotron luminescence microspectroscopy setup on this already well-characterized varnish system, we measured the luminescence emission from micrometric spots of this underlying stratum (Figure 3). The features of a typical emission spectrum using 272 nm excitation are maxima at 340 and 415 nm that were attributed to the luminescence of tryptophan and of collagen cross-links59,60 (Figure 3B). The luminescence feature at 415 nm indicates the presence of collagen, that is of gelatin, in the firstapplied stratum of the varnish system. A typical luminescence emission spectrum of a reference hide glue (Sennelier, Paris) film, collected under similar conditions, presents the same spectral features attributed to tryptophan and collagen cross-links emissions (339 and 411 nm, respectively), as well as an emission band at shorter emission wavelength (maximum at 303 nm), which can be attributed to tyrosine19,61 (Figure 3C). Observation of the latter was impossible in our sample because of the residual contribution from the Rayleigh scattering. This case study indicates that synchrotron luminescence microspectroscopy allows the detection of luminophores in proteins that may be specific to particular proteinaceous materials or of their aging chemical modifications at a micrometric spatial resolution, while preserving the layered structure of the sample. Synchrotron Multispectral Full-Field Luminescence Microscopy. Synchrotron multispectral full-field luminescence microscopy was performed using an Axio Observer Z1 microscope (Carl Zeiss MicroImaging, Jena, Germany) using a 40 Ultrafluar (Zeiss, NA = 0.6) corrected for the 170 μm thick quartz coverslips. The monochromatized radiation is reflected toward the samples by a set of dichroic mirrors (Omega opticals) adapted to the excitation. 2D images were recorded with a 696 520 pixels Peltier-cooled Rolera XR Camera (Qimaging, Surrey, Canada), with 12.9 12.9 μm2 pixel size, and controlled using the Qcapture Pro software (Qimaging). The projected pixel size was calculated at 290 nm from an optical test pattern and corroborated by comparing obtained images with light microscopy images of one sample. The layout of the transfer optics to the microscopes and from the microscopes to the detectors is described in F. Jamme et al.53 In the context of the present work, the setup was further improved by inserting a set of spectral interference band-pass filters in a filter wheel (Prior) between the microscope and the detector. The filters used were centered at 500, 550, 600, 650, and 700 nm (40 nm full-width half-maximum, fwhm, Corion S40, Newport) in the visible range, and at 330 nm (60 nm fwhm, Fx3000, Omega opticals) in the UV. The filters were used to generate six-spectral-band image-cubes. The integration time was optimized for each acquisition and varied between 30 and 180 s. The data were processed using the ENVI software (ITT Visual Information Solutions). We applied the imaging technique to the zinc oxide paint sample previously analyzed by synchrotron luminescence microspectroscopy (see above). We used the emission band-pass filter in the 300-360 nm range (60 nm fwhm) to image the ZnO band 1740
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Figure 3. (A). Transmitted light microscopy image of a 1-μm thick microtomed cross-section of the Boquay cello varnish system. From below: wood cells, underlying stratum, incomplete upper stratum (darker fragments). The red circle indicates the position and size (about 1 μm diameter) of the analysis spot for the spectrum shown in Figure 3B. (B) Emission microspectroscopy spectrum obtained from the underlying (first-applied) stratum (red circle on Figure 3A); 272 nm excitation, 60 s integration time. The emission band tail observed below 320 nm is due to the Rayleigh scattering of the excitation light. (C) Typical emission spectrum obtained on a hide glue film reference sample (hide glue from Sennelier, Paris); 275 nm excitation, 120 s integration time.
Figure 4. Synchrotron multispectral full-field luminescence microscopy images of an aged ZnO paint film in linseed oil. Images were collected at an excitation of 260 nm, in a setup providing a 290 nm projected pixel size. (A) Luminescence imaged using a band-pass filter centered at 500 nm (40 nm fwhm); 63 s integration time for the whole image. (B) Band edge emission imaged using a band-pass filter centered at 330 nm (60 nm fwhm); 40 s integration time for the whole image. The gray levels of images (A) and (B) are correlated to the integration times and to the relative spectral sensitivities of the system (camera/emission filter) in the respective emission ranges imaged. The initial 696 520 pixel images were cropped to 370 520 to display the sample area. The white box indicates the area displayed in Figure 4C. (C) Detail from Figure 4B showing that the signal from individual particles (indicated by arrows) typically of 2 pixels (equivalent to 580 nm) in diameter could be resolved.
edge emission (centered at 383 nm) of the zinc oxide particles (Figure 4B). This image generated is consistent with that obtained by luminescence microspectroscopy (Figure 2B). The broad and less intense feature attributed to a ZnO ‘trap state’
emission with a maximum around 510 nm could be imaged with a 40 nm fwhm emission band-pass filter in the 480-520 nm range (Figure 4A). The band-pass filters chosen for recording of images shown in Figure 4A and 4B did not allow the imaging of the ZnO 1741
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Analytical Chemistry emission centered around 410 nm. The images shown in Figure 4A and 4B also illustrate how the luminescence in the UV and the visible ranges of the ZnO particles (band edge and trap state, respectively) is affected by the absorbance properties of the organic binder and multiple scattering by ZnO particles. Matrix effects can probably explain the differences between the two images: oil absorption is negligible at wavelengths longer than 450 nm; thus, the broad trap state emission centered at 510 nm may come from deep inside the paint film and undergo multiple scattering by ZnO particles, leading to a diffuse emission distribution (Figure 4A). Luminescence of the binding medium may also contribute to this feature. Conversely, the strong absorption of the oil medium at wavelengths shorter than 400-420 nm strongly limits the contribution of the ZnO band edge emission to the image in the 360-400 nm range to the emission of particles located near the paint film surface, therefore producing a sharper image (Figure 4B).
’ DISCUSSION We demonstrate the feasibility and interest of the synchrotron VUV-UV-visible monochromatic beam to study cultural heritage cross-sections nondestructively. In the context of this study the three major aspects of the synchrotron source are (a) the extended wavelength range, (b) the continuous tunability of the source, and (c) its brightness. The wavelength domain that could be attained ranged from 180 to 600 nm. In the VUV region this far exceeds the shortest wavelengths that can be attained with adequate flux using conventional setups based either on xenon sources (typically 250 nm) or commercial tunable laser sources such as OPOs. The tunability of the source over this entire energy domain is provided for both setups by the use of an excitation monochromator with a typical resolving power of 1000. With the microspectroscopy setup, the use of a grating at the emission stage led to a spectral resolution of 0.26 nm. This ensures a high level of monochromaticity that can greatly enhance the selectivity for analysis of compounds bearing luminophores, even when they need to be excited and/or detected in the VUV-UV range. In the indicated working conditions, for both setups, the photon flux was high enough to collect luminescence information at a submicrometric lateral resolution. The current spatial resolutions could be inferred by assessing the possibility to discriminate between adjacent ZnO particles. The spatial resolutions attained were as follows: (1) 400 nm in the microspectroscopy setup achieved by collimating the detected beam (Nyquist criterion with a 800 nm diameter beam size oversampled four times), (2) 590 nm in the full-field microimaging setup with a 40 objective. For the latter, we considered that the spatial resolution was better than twice the projected pixel size reached (i.e., 2 290 = 580 nm) and individual particles from this diameter up could easily be resolved (Figure 4C). For both setups, the brightness of the source was sufficient to allow short collection times on real samples ranging from fractions of a second to 1 min. This duration corresponds respectively to the acquisition of a high spectral resolution spectrum and the collection of 361 920 pixels, for the microspectroscopy and the full-field microscopy setups. We could therefore record at this spatial resolution luminescence spectra of both inorganic and organic materials in cross-section samples by selecting efficient excitation and emission conditions. In particular, signatures specific for intrinsically fluorescent amino acids (such as tryptophan) and for collagen cross-links
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could be measured on historical samples using synchrotron UV microspectroscopy. We suggest that more information on the structure and denaturation of the protein materials in the context of historical samples could be deduced from luminescence microspectroscopy data. For instance, the high sensitivity of the tryptophan emission to its local environment (shifting, relative quenching)61,62 could be exploited to probe structural (folding/ unfolding, etc.) and functional characteristics of protein materials at the submicrometer scale. Also, further developments are required, for instance to assess the changes in the luminescence of protein-based materials at the micrometer-scale in the context of cultural heritage artifacts. In fact, many factors can induce changes in the emission spectra of molecules such as the variability in primary sources of materials, the inconsistency of historical preparative methods, the influence of aging conditions on the formation of degradation products, and the effects from medium and other admixed materials (pigments, extenders, etc.). We also suggest further important developments in the fullfield multispectral imaging of organic binding media in crosssections using their luminescence properties, which may be illustrated from the following example of multispectral full-field luminescence microscopy of a multilayered varnish microsample. The sample is a cross-section from the Provigny violin, one of the most renowned instruments made by Antonio Stradivari (1716, Cremona, collection Musee de la musique, Paris, inv. num. E.1730, Figures 5A and 5B), which was previously extensively studied.13 The two strata numbered 1 and 3 in Figure 5C exhibit very similar FT-IR features and are both oil-based coatings; their interface could not be properly visualized.13 We have acquired multispectral cubes of luminescence images obtained at several excitation wavelengths using the tunable synchrotron light source and a set of narrow band emission filters. The reconstruction of a false-color RGB image from the luminescence image-cube obtained at 340 nm excitation illustrates the enhanced spectral contrast provided by this technique to highlight the interface between the two adjacent varnish strata 1 and 3 (Figure 5C). It is therefore a technique that can be complementary to, and in some cases more specific than, synchrotron FT-IR microspectrometry13 or regular epiluminescence microscopy (Figure 5B) to increase the contrast between thin layers containing organic materials in cross-sections. The particular case of ZnO in paint layers highlights two additional major interests in performing synchrotron UV-visible imaging. First, luminescence microspectroscopy raster-scanning and full-field imaging performed on a ZnO paint sample show that its band edge emission can be used as a marker for its identification and localization. This emission band was previously reported in cultural heritage works, yet it is not a commonly used marker for ZnO.35,41 The characteristic band edge luminescence can be used even at low concentrations and in the presence of additional materials from historical cross-sections at a submicrometric resolution. This result is in agreement with works focused on synthetic ZnO nanostructures using cathodoluminescence.63 In the present work, the setup discussed was shown to be versatile. Indeed, with a synchrotron light source that is continuously tunable from the VUV to the visible, we are able to selectively excite the specific luminescence of compounds in complex mixtures as encountered in archeological and historical materials. Second, we observed that isolated aggregates of ZnO show specific luminescence around 410 nm at the submicrometer scale. Emission from such localized areas cannot be observed using luminescence spectroscopy at the macroscale, as the corresponding signal would be masked by the more intense 1742
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Figure 5. (A) Transmitted light microscopy image of a cross-section of a varnish from the Provigny violin by A. Stradivari, exhibiting several strata of organic binding media materials on top of spruce wood. (B) Epiluminescence microscopy image of the cross-section, using a mercury lamp and a filter cube (dichroic mirror, emission and excitation filters). Excitation light is composed of the 405 and 436 nm mercury lines. Epiluminescence image is recorded for wavelengths above 468 nm using a color CCD camera. (C) Reconstructed RGB full-field luminescence microscopy image of the crosssection; 340 nm excitation. Images collected band-pass emission filters centered at 500, 600, and 700 nm (all with 40 nm fwhm) (respectively 150, 45, and 120 s integration times) have been corrected from dark field and respectively attributed to the blue, green, and red channels. The initial 696 520 pixel images were cropped to 341 485 to display the sample. (D) Uncalibrated luminescence spectra extracted from the full multispectral image-cube at three distinct locations (shown in Figure 5C); 340 nm excitation. Spectra generated by averaging over 5 5 pixel areas. Because of their limited dimensions, the position and size of these areas (dark circles) could not be exactly represented in Figure 5C.
and ubiquitous band edge luminescence signal. This emission is characteristic of defect-induced states in the band gap that could be indicative of manufacturing processes or long-term alteration features of the pigment material. Cocharacterization of the binder by UV-visible, although not pursued here, could be performed, as suggested by the comparison of full-field microscopy images (Figure 4A versus 4B). Indeed, the luminescence of the binder can be selectively induced by using an excitation wavelength above 383 nm, corresponding to energy lower than the band gap energy of ZnO. In this way, the luminescence from ZnO would not be induced and only that of binder would occur. Moreover, it is likely that the luminescence of the binder in the visible range would not be distorted by optical properties of ZnO, which shows a flat and high reflectance factor in this domain. Current possibilities and limitations of the setup developed at the DISCO beamline highlight further methodological developments to be pursued that exploit the synchrotron VUV-UVvisible spectroscopy and imaging capabilities. The synergy of the (X, Y, I, λ) maps collected with the confocal microscope and the image-cubes obtained with the fullfield microscopes leads to new experimental designs to unveil the complexity of heterogeneous, degraded cultural heritage samples. A first analytical sequence consists of using the full-field imager with a set of emission filters to image the sample. Local regions of interest with specific, distinct spectral features are then studied further by high-resolution luminescence microspectroscopy. Following this, the detection chain of the full-field imager is adjusted to map the spectral features determined from localized
high-resolution measurements. Selection of optimal excitation wavelengths and emission filters will allow highly sensitive, rapid imaging of chosen spectral features. The analytical sequence could be accelerated by facilitating the switch between collection of full-field and high-resolution data. While the spectral resolution for full-field emission spectra extracted from an image-cube is currently limited by the number of filters used and their respective band-pass widths, the achievable spectral resolution for excitation is typically 0.2 nm at 200 nm. The ability to retrieve emission and excitation luminescence data for each pixel of the image at the spatial resolution of our setup (one camera pixel corresponds to 290 nm on the sample) can be seen from Figure 5C and 5D and opens the door to the adaptation of a spectroimager to the synchrotron fullfield setup. Such a high-resolution synchrotron full-field spectromicroscopy setup would even further improve the spatial and spectral resolutions and allow the refinement of the luminescence spectral interpretation of historical samples.43 The ultimate goal is to attain diffraction-limited spatial resolution while working at a high emission spectral resolution, comparable to that obtained from implementation of the luminescence microspectroscopy setup. The spatial registration of the images composing each imagecube collected with the full-field imaging setup will allow the spatial resolution to be maintained from individual images to the cube. The registered image-cube can then be accurately radiometrically calibrated, either based on calibrated luminescence microspectroscopy results or based on instrumental parameters 1743
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Analytical Chemistry (incoming flux distribution, filters and objective transmission characteristics, detector spectral sensitivity, etc.) in order to provide luminescence spectra for comparison to reference data.43 A complementary direction is the statistical segmentation of the multispectral image for a better discrimination of materials. Attaining a high resolution in luminescence spectroimaging is therefore a key factor for decreasing the complexity of the mixtures of species contributing to each pixel, thereby obtaining more efficient data segmentation. For both setups, FLIM capabilities could be implemented. On account of the pulsed nature of the synchrotron source, in turn connected to the electron bunch characteristics determined by the synchrotron storage ring operation mode, heterodyne phase-sensitive detection could be implemented.64,65 This would allow even more specific discrimination between different compounds based on the rate of decay of their luminescence. In particular, measuring the sensitivity of the luminescence lifetime to the local chemical environment would be of strong interest for determining the distribution of degradation products found in conservation science studies of cultural and archeological artifacts.42
’ CONCLUSION Synchrotron VUV-UV-visible luminescence has been demonstrated to be a novel, powerful, nondestructive technique for chemical analysis to characterize and identify inorganic and organic compounds within complex mixtures in cross-sections and other small samples of interest to cultural heritage. Until now, the energy ranges of synchrotron radiation used to study cultural heritage artifacts were primarily in the X-ray and infrared spectral domains.22,66-68 Here, we have shown that new developments based on synchrotron VUV-UV-visible light provide an efficient probe to study, at a high lateral resolution, small quantities of compounds encountered in art and archeology. Ranges of art and archeological materials exhibit specific luminescence features that can be measured and imaged with precision including binding media, organic dyes, gums and resins, polymers used as consolidating agents, degradation products, etc., that may be present in trace amounts or limited spatial distribution. The method can, therefore, find its place in the analytical sequence used to study heritage materials. The microspectroscopy setup implemented provided high spatial and emission spectral resolutions, while the multispectral full-field imager provided high spatial resolution and short collection times. Preliminary measurements regarding the interest and the feasibility to implement a full-field spectroimager are reported. Research on historical materials motivates further development of available synchrotron VUV-UV-visible spectroscopy and imaging techniques including the optimal conjugation between spot analyses and full-field imaging, the implementation of fluorescence lifetime imaging, and an easier collection of fullfield images in successive emission wavelength ranges. Such developments could also more largely benefit the broader scientific community working on complex heterogeneous samples encountered in environmental, materials, and biological sciences.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
ARTICLE
’ ACKNOWLEDGMENT We thank Balthazar Soulier for providing the varnish sample from the cello by J. Boquay, E. Rene de la Rie, and John K. Delaney (National Gallery of Art, Washington D.C) for providing, respectively, the zinc oxide paint sample and the set of emission filters, Serge Cohen (IPANEMA), and synchrotron SOLEIL for beamtime, under project no. 20090761. The IPANEMA platform is jointly developed by CNRS, MNHN, the French Ministry of Culture and Communication, and SOLEIL and benefits from a CPER grant (MESR, Region ^Ile-de-France).69 M.T., J.-P.E., and L.B. contributed equally to this work. ’ REFERENCES (1) Cotte, M.; Susini, J.; Sole, V. A.; Taniguchi, Y.; Chillida, J.; Checroun, E.; Walter, P. J. Anal. At. Spectrom. 2008, 23, 820–828. (2) Bartoll, J.; Hahn, O.; Schade, U. Stud. Conserv. 2008, 53, 1–8. (3) van der Weerd, J.; Heeren, R. M. A.; Boon, J. J. Stud. Conserv. 2004, 49, 193–210. (4) Joseph, E.; Prati, S.; Sciutto, G.; Ioele, M.; Santopadre, P.; Mazzeo, R. Anal. Bioanal. Chem. 2010, 396, 899–910. (5) Prati, S.; Joseph, E.; Sciutto, G. Acc. Chem. Res. 2010, 43, 792–8014. (6) Mazel, V.; Richardin, P.; Touboul, D.; Brunelle, A.; Walter, P.; Laprevote, O. Anal. Chim. Acta 2006, 570, 34–40. (7) Mayet, C.; Dazzi, A.; Prazeres, R.; Allot, F.; Glotin, F.; Ortega, J. M. Opt. Lett. 2008, 33, 1611–1613. (8) Sciutto, G.; Dolci, L. S.; Buragina, A.; Prati, S.; Guardigli, M.; Mazzeo, R.; Roda, A. Anal. Bioanal. Chem. 2010, DOI 10.1007/s00216010-4258-7. (9) Cartechini, L.; Vagnini, M.; Palmieri, M.; Pitzurra, L.; Mello, T.; Mazurek, J.; Chiari, G. Acc. Chem. Res. 2010, 43, 867–876. (10) Lehmann, J.; Solomon, D.; Kinyangi, J.; Dathe, L.; Wirick, S.; Jacobsen, C. Nat. Geosci. 2008, 1, 238–242. (11) Jacobsen, C.; Flynn, G.; Wirick, S.; Zimba, C. J. Microsc. 2000, 197, 173–184. (12) Echard, J.-P.; Cotte, M.; Dooryhee, E.; Bertrand, L. Appl. Phys. A: Mater. Sci. Process. 2008, 92, 77–81. (13) Echard, J.-P.; Bertrand, L.; von Bohlen, A.; Le H^ o, A.-S.; Paris, C.; Bellot-Gurlet, L.; Soulier, B.; Lattuati-Derieux, A.; Thao, S.; Robinet, L.; Lavedrine, B.; Vaiedelich, S. Angew. Chem., Int. Ed. 2010, 49, 197–201. (14) Bertrand, L.; Robinet, L.; Cohen, S. X.; Sandt, C.; Le H^o, A.-S.; Soulier, B.; Lattuati-Derieux, A.; Echard, J.-P. Anal. Bioanal. Chem. 2010, DOI: 10.1007/s00216-010-4288-1. (15) Daher, C.; Paris, C.; Le H^o, A.-S.; Bellot-Gurlet, L.; Echard, J.-P. J. Raman Spectrosc. 2010, 41, 1204–1209. (16) Thoury, M.; Elias, M.; Frigerio, J.-M.; Barthou, C. Appl. Spectrosc. 2007, 61, 1275–1282. (17) Nevin, A.; Echard, J.-P.; Thoury, M.; Comelli, D.; Valentini, G.; Cubeddu, R. Talanta 2009, 80, 286–293. (18) Nevin, A.; Comelli, D.; Valentini, G.; Cubeddu, R. Anal. Chem. 2009, 81, 1784–1791. (19) Nevin, A.; Anglos, D.; Cather, S.; Burnstock, A. Appl. Phys. A: Mater. Sci. Process. 2008, 92, 69–76. (20) Salvado, N.; Butı, S.; Tobin, M. J.; Pantos, E.; Prag, A. J. N. W.; Pradell, T. Anal. Chem. 2005, 77, 3444–3451. (21) Van der Snickt, G.; Dik, J.; Cotte, M.; Janssens, K.; Jaroszewicz, J.; De Nolf, W.; Groenewegen, J.; Van der Loeff, L. Anal. Chem. 2009, 81, 2600–2610. (22) Cotte, M.; Dumas, P.; Taniguchi, Y.; Checroun, E.; Walter, P.; Susini, J. C. R. Phys. 2009, 10, 590–600. (23) Rorimer, J. J. Ultra-Violet Rays and Their Use in the Examination of Works of Art; Metropolitan Museum of Art: New York, 1931. (24) Eibner, A. Mouseion 1933, 21-22, 32–68. (25) Eibner, A. Angew. Chem. 1932, 45, 301–307. (26) Eibner, A. Chem. Ztg. 1931, 593–604; 614–615; 635–637; 655– 656. 1744
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