Fluorescence Lifetime Imaging and Fourier Transform Infrared

Fluorescence Lifetime Imaging and Fourier Transform Infrared Spectroscopy of Michelangelo’s David DANIELA COMELLI,* GIANLUCA VALENTINI, RINALDO CUBEDDU, and LUCIA TONIOLO INFM-Dipartimento di Fisica and CEQSE-CNR, Politecnico di Milano, Piazza Leonardo da Vinci 32, I-20133, Milan, Italy (D.C., G.V., R.C.); and Istituto per la Conservazione e la Valorizzazione dei Beni Culturali—CNR Sezione di Milano ‘‘Gino Bozza’’— Politecnico di Milano, Piazza Leonardo da Vinci 32, I-20133, Milan, Italy (L.T.)

We developed a combined procedure for the analysis of works of art based on a portable system for fluorescence imaging integrated with analytical measurements on microsamples. The method allows us to localize and identify organic and inorganic compounds present on the surface of artworks. The fluorescence apparatus measures the temporal and spectral features of the fluorescence emission, excited by ultraviolet (UV) laser pulses. The kinetic of the emission is studied through a fluorescence lifetime imaging system, while an optical multichannel analyzer measures the fluorescence spectra of selected points. The chemical characterization of the compounds present on the artistic surfaces is then performed by means of analytical measurements on microsamples collected with the assistance of the fluorescence maps. The previous concepts have been successfully applied to study the contaminants on the surface of Michelangelo’s David. The fluorescence analysis combined with Fourier transform infrared (FT-IR) measurements revealed the presence of beeswax, which permeates most of the statue surface, and calcium oxalate deposits mainly arranged in vertical patterns and related to rain washing. Index Headings: UV fluorescence; Time-resolved spectroscopy; Imaging; Fourier transform infrared spectroscopy; FT-IR spectroscopy; Marble sculptures.

INTRODUCTION In the field of artwork conservation and care, nondestructive analyses performed in situ have gained increasing importance in the last years because they provide valuable information in real time on the materials present on artifacts. Such materials can be original materials used by the artist, new formation materials deriving from deterioration processes, and restoration materials that have been applied on the surfaces throughout the centuries. Among the nondestructive investigations that have been applied in situ, diffuse reflectance,1–3 either in visible and near-infrared spectral bands, and several spectroscopic techniques, like optical fluorescence,1,4 X-ray fluorescence,5,6 and vibrational spectroscopy,6,7 are the most attractive. Laser-induced breakdown spectroscopy has also been applied to the analysis of works of art; nevertheless, this technique is just minimally invasive, since a microscopic portion of the object is destroyed.3,8 Aside from the physical parameters that are specifically measured by any technique, a very important classification splits the investigation methods into imaging methods, such as diffuse reflectance and radiographic recording, and non-imaging methods, such as most of the spectroscopic measurements. The compositional heterogeneReceived 11 January 2005; accepted 9 June 2005. * Author to whom correspondence should be sent.

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ity of any artwork, which is very often an important part of the author’s message, makes imaging techniques more suitable than point measurements to understand the composition of works of art, since they preserve the morphology of the object. On the other hand, point measurements usually provide a richer data set (e.g., a high-resolution spectrum), while imaging methods very often give only an intensity map in a specific spectral band. Fluorescence lifetime imaging (FLIM) is a technique that measures at once the amplitude and the decay time maps of the fluorescence emission of a sample after excitation with very short pulses of ultraviolet (UV) light. FLIM has been successfully applied to several fields, including combustion analysis,9 fluorescence microscopy,10 and medical diagnosis.11,12 In the field of artwork conservation, it gives an immediate perception of the artwork morphology, together with functional information. In fact, the amplitude map mainly depends on the emission intensity and shows the artwork shape, while the lifetime map is correlated with its compositional heterogeneity, since any compound usually exhibits a characteristic fluorescence lifetime. Ultraviolet-induced fluorescence is specially suited to the study of organic compounds, which are largely present in works of art; yet, inorganic contaminants can also be revealed through an indirect effect on the background fluorescence emission. Notwithstanding its effectiveness, optical fluorescence cannot provide an exhaustive characterization of a sample alone, since the emission is typically due to a mixture of several chromophores. Moreover, the molecular environment experienced by chromophores, which in turn might change with age, conservation status, etc., often influences the properties of the emission.13 Thus, the extraction of analytical information from fluorescence measurements is not straightforward, but requires the support of other measurements. However, FLIM is a powerful tool to distinguish between regions showing different compounds, which can be discriminated on the basis of their emission lifetime. Starting from these considerations, we developed a portable system for advanced fluorescence imaging and spectroscopy and we applied it to the analysis of surfaces of artistic interest. The system is made of two main units: a FLIM device and a spectroscopic unit based on an optical multichannel analyzer (OMA). The information obtained with fluorescence measurements indicates the points on the artwork at which to take microsamples to be analyzed in the laboratory by means of techniques

0003-7028 / 05 / 5909-1174$2.00 / 0 q 2005 Society for Applied Spectroscopy

APPLIED SPECTROSCOPY

such as optical microscopy and Fourier transform infrared (FT-IR) spectroscopy. Analytical measurements give a synergic effect with fluorescence imaging: in fact, the chemical identification of the materials provided by analytical techniques can be transferred to the whole extension of the artwork thanks to the imaging capabilities of the FLIM apparatus, without the need for extensive microsampling. This measurement protocol has been previously applied to assess the conservation status of renaissance fresco paintings in Castiglione Olona.14 This experience, matured through the study of mural paintings, suggested to us the application of the combination of FLIM and FTIR techniques to investigate the contaminants on the surface of Michelangelo’s David within a diagnostic program aimed at supporting the conservative work carried out in 2004. The present paper reports on the most significant results achieved in this measurement campaign.

imen. Nevertheless, even in the case of multi-exponential behavior of the fluorescence emission, a linear fit performed on the logarithm of acquired data allows the reconstruction of an effective lifetime.15 The lifetime map yields strong contrast for the discrimination of different compounds, as has also been shown by other research groups.16 Further, the processing of a high-resolution image with a linear fitting algorithm requires only a few seconds. Therefore, such algorithms would also be suitable for real-time data processing. Assuming a mono-exponential behavior of the fluorescent emission f , with effective lifetime t and amplitude A: f (t) 5 A exp(2t/t)

the fluence H acquired in each pixel as a function of the delay d, is given by the following formula: H(d) 5 C

MATERIALS AND METHODS Fluorescence Experimental Setup. The fluorescence imaging system used for this study is similar to the one described in Cubeddu et al.11 It is based on a time-gated intensified charge-coupled device (ICCD) (ICCD225, Photek, St Leonards-on-Sea, England) exhibiting a minimum gate width of 5 ns. A sequence of images is acquired by activating the gate of the image detector at different delays with respect to the excitation pulses. In this way, the temporal behavior of the fluorescence is recorded in each pixel. Then, by applying a suitable fitting procedure, which will be described in the following subsection, the fluorescence lifetime map of the field of view is reconstructed. The UV (l 5 337 nm) excitation light is provided by a nitrogen laser (LN203C Laser Photonic, Orlando, FL) that generates 1 ns pulses. The excitation beam is coupled to a silica fiber and delivered to the sample. A homemade trigger circuit and a precision delay generator (DG535, Stanford Research Systems, Sunnyvale, CA) allow the temporal sampling of the emitted fluorescence. The whole system has been assembled in a portable rack, except the gated camera, which is connected to the control unit through a 10 m cable for remote access to any part of the sculpture. An optical multichannel analyzer (OMA EG&G Princeton Applied Research, Princeton, NJ) completes the experimental apparatus. It measures fluorescence spectra from 400 to 800 nm, with a spectral resolution of 1 nm. A second nitrogen laser (VSL-337ND-S, Laser Science Inc.) provides the excitation light. The laser beam is coupled to a silica fiber bundle, which is put in gentle contact with the sample through a metallic spacer covered with a Teflon ring. The bundle is made of a central fiber, which delivers the excitation light to an area 3 mm in diameter, and 20 fibers arranged in two circular rings, which collect the emitted fluorescence. The spacer maintains the fibers at a suitable distance from the sample in order to optimize the superposition of the excited area with the field of view of the collection fibers. Data Analysis. Fluorescent systems can hardly be modeled as mono-exponential due to the simultaneous presence of several emitting molecules in the same spec-

(1)

E

d1w

f (t) dt

d

[ [

1 t2 2 exp 12 t 2 w d 5 CAt 1 2 exp 12 2 exp12 2 t t 5 CAt exp 2

d1w

d

]

]

(2)

where w is the gate width and C is a constant dependent on the efficiency of the detection system. The effective lifetime t and the amplitude A can be reconstructed by a least mean squares fit performed on N time samples, leading to the following equations:

O d 2 1O d 2 t52 N O d ln H(d ) 2 O d O ln H(d ) H(d ) d w A 5 O H(d ) exp1 2 1 2 exp 12 2 t t [ t ] 2

2 k

N

k

k

k

k

k

k

k

k

(3)

k

k

21

k

k

k

(4)

k

Both t and A are matrices that represent the spatial maps of the fluorescence lifetime and amplitude of the sample in the field of view of the gated camera. As far as the meaning of the two matrices t(x, y) and A(x, y), the first reveals areas with different chemical composition, while the second gives information on the relative abundance of the fluorescent materials in the field of view. By merging the two maps, a third one, named the HSV map, is created. This map is based on the HSV (Hue, Saturation, and Value) color model.17 The luminance (value) of each pixel is proportional to the fluorescence amplitude, while the hue represents the lifetime, keeping the saturation constant at 0.8. In this way, the HSV map allows one to easily associate the functional information provided by the lifetime (hue) to the morphology of the analyzed region, given by the fluorescence amplitude (value). Experimental Procedure. For the measurements on David, the gate of the image detector was set to 100 ns, wide enough to get almost all the fluorescent light emitted by the sculpture. A set of 12 images was recorded, corresponding to delays of 0, 2, 3, 5, 8, 10, 12, 15, 20, APPLIED SPECTROSCOPY

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30, 40, and 50 ns with respect to excitation pulses. Then, the images were processed in order to calculate the FLIM maps. From the reconstructed maps, it was possible to distinguish regions showing similar characteristics, as will be described hereafter. In correspondence with each homogeneous region, a fluorescence spectrum was recorded and, at a few selected points, a microsample of the material superimposed on the sculpture was taken with a scalpel and the aid of a portable microscope; only materials overlaying the marble surface were sampled; no sampling of the sculpture itself was allowed. Microsamples were examined with a Fourier transform infrared spectrophotometer (Nicolet Nexus) coupled to an FT-IR microscope (Nicolet Continuum). The detector was a liquid N2 cooled HgCdTe detector. Spectra were recorded using a Graseby–Specac diamond cell accessory in transmission mode between 4000 and 700 cm21. RESULTS AND DISCUSSION

FIG. 1. Fluorescence spectra normalized to the peak value of: (a) a quarry sample of Carrara marble; (b) the David’s surface close to the right ear lobe; and (c) the inner surface of a small David’s fragment. The Carrara marble sample shows a fluorescence emission peaking at a lower wavelength with respect to the emission coming from the David’s marble.

An extensive measurement campaign was carried out over the David’s entire surface. A total of 45 fluorescence images were collected in areas representative of conservation status and geometrical orientation, which could be relevant for the accumulation of deposits or for the action of atmospheric agents. To this purpose, we considered areas with different surface roughness or with different slope with respect to the vertical direction. Also, the visual inspection and a set of photographs taken with a Wood lamp indicated several areas of interest, e.g., areas showing a yellowish color in white light or bright spots in UV light. A common finding that was observed in all the areas considered in this study was the unexpected intense emission coming from the David’s surface in response to UV excitation light. It is important to emphasize that minerals, excited by UV light, often emit a characteristic luminescence; the emission is due to impurities located inside the crystal lattice of the material. In marble, for example, Mn 21 can substitute for Ca 21 ions, becoming the main emitting centers, whereas Sm31 and Dy31 are minor defects. Yet, studies on calcite, of which marble is made, have shown that these types of impurities are characterized by a luminescence having a decay time on the order of microseconds,18,19 thus non-detectable with our fluorescence setup, which works on the nanosecond time scale. Furthermore, Carrara marble has a very low concentration of Mn 21 ions. To confirm the low fluorescence expected from calcium carbonate, a quarry sample of Carrara marble was measured in our laboratory with the same experimental setup used to analyze the Michelangelo masterpiece. The emission from the quarry sample was on average 2–3 times lower than that from the David and showed a peak around 500 nm, while the peak of the David’s emission is always beyond 500 nm and sometimes is even beyond 550 nm. Figure 1 shows three normalized spectra that well-depict this concept: spectrum a refers to the sample of quarry Carrara marble, spectrum b was taken from the David’s surface close to the right ear lobe, and spectrum c was measured from a small fragment of the David, a few millimeters thick, collected from the second toe of the

left foot after a vandalism carried out in 1991. This last spectrum is especially interesting since it was taken from the inner surface of the fragment. The differences between spectrum a and spectra b and c are evident, mainly concerning the peak position. Also, the fluorescence lifetime easily distinguishes the Carrara quarry sample from the David’s marble. In fact, even if the time behavior of both emissions can hardly be modeled by a mono-exponential decay, the effective lifetime of Carrara marble fluorescence is definitely longer (.7–8 ns) than that of the David’s marble by at least 2 ns. Hence, the differences in spectral features, temporal behavior, and intensity between the fluorescence of Carrara marble and that of David’s marble let us suppose that David’s surface is extensively permeated by organic materials, which have their own emission properties and determine the fluorescence features of the statue much more than the impurities, naturally occurring in the marble microstructure, can do. This observation can be explained as the result of centuries of environmental contamination and as the consequence of several conservation treatments carried out in the past. In the following, some details exemplificative of the organic materials and inorganic salts more frequently found on the David’s surface will be discussed. Some small fluorescent spots, sporadically present on the surface, e.g., on the right forearm, on the back, on the shoulders, and on the trunk, have a bright emission and appear light blue under the Wood lamp. They have a fluorescence lifetime greater than 6 ns (Fig. 2) and a spectral peak around 520 nm. Such spots are well outlined both in the amplitude and in the lifetime maps and were identified as composed of beeswax through the FTIR analysis 20 (Fig. 3 and Table I) of a microsample collected in the back of the statue, on the left of the sling. Since the fluorescence decay time of the drops of beeswax is the longest ever found on the statue, using the lifetime maps collected all over the marble surface, it was possible to localize most of such wax remains. The presence of large quantities of wax in the small dips that characterize the marble finishing of the sling, located on the back of the statue (data not shown), confirms that the

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FIG. 3. FT-IR spectrum of a microsample collected on the back of the sculpture; absorption peaks of beeswax (2955, 2918, 2850, 1742, 1468, 1379, 1175, 1100, and 722 cm21) and whewellite (calcium oxalate monohydrate, 1631, 1316, and 780 cm21) are evident.

istic gray color and simply act as absorbers for the excitation light. On the contrary, the lifetime only depends on the chemical structure of the fluorophores uniformly adsorbed into the crystalline structure. Figure 5 shows the emission spectrum of the left hand compared with the spectrum taken in one of the fluorescent spots shown in Fig. 3. The lifetime (6 ns) and the spectrum of the emission taken in the left hand are similar to those of wax, confirming that the material distributed all over the sculpture is very likely based on this organic compound. Wide regions of the statue present a vertical pattern made of irregular stripes that can hardly be perceived in TABLE I. Infrared frequencies of minerals and organic compounds of the collected spectra. Compound/mineral FIG. 2. Fluorescence analysis performed on the right forearm of the David: (a) color picture of the area; (b) fluorescence lifetime map; and (c) fluorescence amplitude map. The maps show the presence of fluorescent spots on the marble surface characterized by a lifetime close to 6 ns.

whole David’s surface underwent a conservation treatment based on beeswax in 1813, as can be inferred from archive documents. 23 Such treatment, called ‘‘encausto’’, was carried out to protect the statue from rainfall and other atmospheric precipitations. According to the recipe reported in ancient treatises, the encausto was based on hot wax; after about 200 years, not withstanding several cleanings, wax is still widely present on the statue, as evidenced by FLIM measurements combined with FT-IR spectroscopy. As a further example of these findings, Fig. 4 shows the lifetime (Fig. 4b) and the amplitude (Fig. 4c) of the back of the David’s left hand. The lifetime map is rather uniform, while the amplitude map presents a pattern that is reminiscent of the typical marble veins, also visible in the white-light image (Fig. 4a). Actually, the amplitude map accounts for absorbance differences due to the presence of metal cations that give the marble its character-

a

Beeswax

Whewellite (calcium oxalate monohydrate)a,b Gypsum (calcium sulfate dihydrate)b

Calcite (calcium carbonate)b Quartz (silicium dioxide)b a b

Frequency (cm21) 2955 2918 2850 1742 1468 1379 1175 1100 722 1631 1316 780 3543 3407 1683 1621 1128 672 1798 1442 875 713 1080 798 780

Vibration stretch. nas C–H stretch. nas CH2 stretch. ns CH2 stretch. ns C5O sciss. ds CH2 bend. sym. CH3 wag. CH2 stretch. ns C–C rock. CH2 stretch. nas CO22 stretch. ns CO22 stretch. n –OH (H2O) stretch. n –OH (H2O) bend. H–O–H bend. H–O–H stretch. SO422 stretch. nas CO322 stretch. ns CO322 stretch. nas Si–O

Ref. 21. Ref. 22.


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FIG. 4. Fluorescence analysis performed on the back of the David’s left hand: (a) color picture of the area; (b) fluorescence lifetime map; and (c) fluorescence amplitude map. The lifetime map shows a uniform decay time close to 6 ns, suggesting the presence of wax adsorbed by the marble.

FIG. 5. Fluorescence spectra normalized to the peak value taken (a) on the back of David’s left hand and (b) on a fluorescence spot on the right forearm. The similarity of the emissions suggests that the two surfaces are permeated by the same compound.

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FIG. 6. Fluorescence analysis performed on the back of the left thigh: (a) color picture of the area; (b) fluorescence lifetime map; and (c) fluorescence amplitude map. In the fluorescence lifetime map it is possible to observe a vertical stripe characterized by a short lifetime value, close to 4 ns. Very likely, in this region the fluorescence emission of the marble, mostly due to the underlying wax residues, is dumped by the presence of inorganic salts and metal cations.

white light and that give a dark brown emission on inspection under the Wood’s lamp. The fluorescence maps of these details present low amplitude and short lifetime (around 4–5 ns), whereas those parts of the surface that appear clean and healthy have a longer lifetime ($6 ns), similar to the one measured in the left hand. Figure 6 shows an example of such a region, located in the rear part of the left thigh. Upon visual inspection, the morphology of the short lifetime sectors can be correlated to surface erosion where salts and particulate matter are present. The FT-IR spectrum (Fig. 7 and Table I) of the brownish deposits established that they generally contain gypsum (calcium sulfate dihydrate), along with weddelite or whewellite (calcium oxalate dihydrate or monohydrate), calcite (calcium carbonate), and quartz (silicon dioxide).

FIG. 7. FT-IR spectrum of the brownish deposits; absorption peaks of gypsum (3543, 3407, 1683, 1621, 1128, and 672 cm21) are prevalent; small absorptions of calcium carbonate (1442 cm21), weddellite (calcium oxalate dehydrate, 1324 cm21), and quartz (1001, 798, and 780 cm21) are also visible.

This surface alteration should be ascribed to the outdoor exposition of David sculpture until 1873, when the statue was placed inside the museum ‘‘Galleria dell’ Accademia’’. Deposits or surface patinas of this type (a compact mixture of different minerals often containing small amounts of organic compounds) have been formed over the centuries. 23,24 A complete characterization of the patina could not be achieved since the amount of material we were allowed to take from the statue was just enough to perform vibrational spectroscopy, while gas chromatography and mass spectrometry, which are more suited to the study of the organic fraction of the patina, were precluded. Nevertheless, some insight can be gained from fluorescence images. In fact, in correspondence with the patina, the fluorescence of the marble, mostly due to the underlying wax residues, is decreased to shorter lifetime and lower amplitude. While the decrease in amplitude can be easily explained by the shielding effect of the patina in the outermost surface, the change in lifetime can lead to different interpretations. It can possibly be ascribed either to the presence in the patina of a low amount of fluorescent compounds with a lifetime shorter than that of the beeswax or to some kind of interaction between the beeswax and the inorganic salts of the patina, leading to an increase in relaxation pathways of the excited states. Finally, Fig. 8 shows the HSV map of the lower part of David’s face. A small red spot under the right nostril presents the typical lifetime of wax residues (.6 ns), while the larger yellow spot above the lips has a slightly shorter lifetime (5.8 ns). The two contaminants also greatly differ in spectral features (Fig. 8c). In fact, the spectrum of the spot under the nostril corresponds to that of beeswax, as expected from its lifetime, while the spectrum of the spot over the lip peaks at a definitely longer wavelength. The appearance of this spot led us to suppose that it should be made of organic material, but it was not possible to assess its chemical structure, since microsampling from the David’s face was not permitted. The analysis of many other details of the David’s surface allow us to conclude that three main types of overlaid materials were largely mapped by fluorescence im-

FIG. 8. Fluorescence analysis performed on the lower part of David’s face: (a) color picture of the area; (b) HSV map; and (c) fluorescence spectra showing the different nature of the spots over the lips and under the nostrils.

aging: (1) beeswax residues concentrated in small drops or permeated into the marble surface; (2) salt deposits, mainly composed of gypsum, calcium oxalates, and particulate matter; and (3) some organic contaminants, not precisely identified, located in small areas. The FLIM apparatus, along with other devices, was also tested to compare different cleaning methods applied to small test areas on the statue. As an example, cleaning tests were performed in a region located on the left shin, characterized by the presence of inorganic deposits (mainly composed of gypsum). Figure 9a shows two patches that were treated with different cleaning procedures: the upper patch (G1) was cleaned with a deionized water poultice, while the lower patch (G2) was cleaned with ion exchange resin (DES90). The fluorescence lifetime maps of the two areas taken before (Fig. 9b) and after (Fig. 9c) the cleaning are also shown. The increase in the fluorescence lifetime that takes place after the cleaning (red shift of the false color map) APPLIED SPECTROSCOPY

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CONCLUSION

FIG. 9. Fluorescence analysis performed during a cleaning test performed on the left shin: (a) color picture of the area; and fluorescence lifetime map (b) before and (c) after the cleaning. The increase in the fluorescence lifetime after the cleaning indicates that some inorganic deposits were removed by the treatment.

in both patches indicates that some inorganic deposits have actually been removed by the cleaning procedures. In fact, the reduction in fluorescence dumping associated with the very superficial layer let the long-lived emission of beeswax absorbed in the marble microstructure become more relevant for the calculation of the effective lifetime. Moreover, the slightly greater increase in the lifetime shown by the G1 patch indicates that the water poultice is possibly more effective than ion exchange resin. 1180


The analysis of David by FLIM was a challenging task for our research unit. Michelangelo’s masterpiece is astonishing for its size: it is more than 5 m tall and its surface area is about 20 m 2. Such a large area can be conveniently mapped only with a wide-field imaging system. On the other hand, the field of view of our FLIM device is very often restricted to a few tens of centimeters by the emission intensity of the sample, which is excited by a low-power laser source. Marble is made of calcite grains that, being constituted of CaCO3, should not fluoresce when irradiated with UV light at 337 nm. Yet impurities always present in marble, beyond a well-known luminescence, also give it a faint fluorescence peaking at 500 nm, as we found with our sample of Carrara marble. If the David fluorescence was to be ascribed only to the bulk material, such a faint emission could have been hardly detected with our system. Unexpectedly, the fluorescence signal was strong enough to allow us to take wide images of the statue from a distance of 0.5–1 m, which is the minimum required to approach the David under safe conditions and for a reasonable mapping of such a wide surface. This result has been interpreted assuming that a large amount of organic material is absorbed into the marble. This finding is possibly a general condition of all ancient marble sculptures having a long history of outdoor exposition and conservation practice. Our experience with another sculpture of Michelangelo’s (Pieta` Rondanini, hosted in the museum ‘‘Civiche Raccolte del Castello Sforzesco’’ in Milan) seems to confirm this hypothesis. The widespread presence of organic contaminants on marble sculptures gives our technique a great relevance among the noninvasive diagnostic procedures. The measurement campaign with David showed that the synergic combination of FLIM with spectroscopy and other laboratory measurements on microsamples (such as FT-IR examination) allowed us to identify some of the overlapped materials. This is especially true for beeswax, which has been found all over the statue as a background signal and in well-outlined spots, sometimes looking like drops. Also, most of the inorganic deposits were mapped thanks to the decrease in the lifetime of the underlying fluorescence emission. Fluorescence lifetime imaging has already been successfully applied to the analysis of fresco paintings,14 while the investigation of oil paintings and other paintings is in progress. The results achieved up to now let us suppose that a well-designed investigation protocol based on FLIM, combined with analytical techniques, could be profitably applied to many other fields of conservation, including ancient manuscripts and other artifacts. ACKNOWLEDGMENTS The authors wish to thank the Director of the Galleria dell’ Accademia, Dr. Franca Falletti, and the scientific team coordinator, Dr. Mauro Matteini, for their valuable and constant support during the measurements and the analysis of data. Particular thanks is due to Dr. A. Aldrovandi for the valuable collaboration and the availability of the photographic materials. 1. S. Daniilia, S. Sotiropoulou, D. Bikiaris, C. Salpistis, G. Karagiannis, Y. Chryssoulakis, B. A. Price, and J. H. Carlson, J. Cult. Heritage 1, 91 (2000).

2. C. Balas, V. Papadakis, N. Papadakis, A. Papadakis, E. Vazgiouraki, and G. Themelis, J. Cult. Heritage 4, 330s (2003). 3. K. Melessanaki, V. Papadakis, C. Balas, and D. Anglos, Spectrochim. Acta, Part B 56, 2337 (2001). 4. P. Weibring, T. Johansson, H. Edner, S. Svanberg, B. Sundner, V. Raimondi, G. Cecchi, and L. Pantani, Appl. Opt. 40, 6111 (2001). 5. C. Fiorini and A. Longoni, Rev. Sci. Instrum. 69, 1523 (1998). 6. C. Ricci, I. Borgia, B. G. Brunetti, C. Miliani, A. Sgamellotti, C. Seccaroni, and P. Passalacqua, J. Raman Spectrosc. 35, 616 (2004). 7. M. Bacci, M. Fabbri, M. Picollo, and S. Porcinai, Anal. Chim. Acta 446, 15 (2001). 8. D. Anglos, Appl. Spectrosc. 55, 186A (2001). 9. T. Ni and L. A. Melton, Appl. Spectrosc. 50, 1112 (1996). 10. A. Periasamy, P. Wodnicki, X. F. Wang, S. Kwon, G. Gordon, and B. Herman, Rev. Sci. Instrum. 67, 3722 (1996). 11. R. Cubeddu, A. Pifferi, A. Torricelli, G. Valentini, F. Rinaldi, and E. Sorbellini, IEEE J. Sel. Top. Quantum Electron. 5, 923 (1999). 12. P. J. Tadrous, J. Siegel, P. M. W. French, S. Shousha, E. Lalani, and G. W. H. Stamp, J. Pathology 199, 309 (2003). 13. J. R. Lakowicz, ‘‘Principles of Fluorescence Spectroscopy’’ (Kluwer Academic, New York, 1999), 2nd ed. 14. D. Comelli, C. D’Andrea, G. Valentini, R. Cubeddu, C. Colombo, and L. Toniolo, Appl. Opt. 43, 2175 (2004).

15. R. Cubeddu, D. Comelli, C. D’Andrea, P. Taroni, and G. Valentini, J. Phys. D: Appl. Phys. 35, R61 (2002). 16. K. C. B. Lee, J. Siegel, S. E. D. Webb, S. Leveque-Fort, M. J. Cole, R. Jones, K. Dowling, M. J. Lever, and P. M. W. French, Biophys. J. 81, 1265 (2001). 17. W. K. Pratt, Digital Image Processing (John Wiley and Sons, New York, 1978), p. 32. 18. M. Gaft, R. Reisfeld, G. Panczer, P. Blank, and G. Boulon, Spectrochim. Acta, Part A 54, 2163 (1998). 19. M. Gaft, G. Panczer, R. Reisfeld, and E. Uspensky, Phys. Chem. Minerals 28, 347 (2001). 20. H. Kuhn, Studies Conservation 5, 71 (1960). 21. D. L. Vien, N. B. Colthup, W. G. Fateley, and J. G. Grasselli, The Handbook of Infrared and Raman Characteristic Frequencies of Organic Molecules (Academic Press, New York, 1991). 22. The Stadtler Infrared Spectra Handbook of Minerals and Clays, J. R. Ferraro, Ed. (Stadtler Research Laboratories, Philadelphia, PA, 1982). 23. F. Falletti, ‘‘Historical Research on the David’s State of Conservation’’, in Exploring David: Diagnostic Tests and State of Conservation, S. Bracci, F. Falletti, M. Matteini, and R. Scopigno, Eds. (Giunti Editore, Florence-Milan, Italy, 2004), p. 55. 24. L. Rampazzi, A. Andreotti, I. Bonaduce, M. P. Colombini, C. Colombo, and L. Toniolo, Talanta 63, 967 (2004).


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