1174 Volume 59, Number 9, 2005 APPLIED SPECTROSCOPY0003-7028 / 05 / 5909-1174$2.00 / 0q 2005 Society for Applied Spectroscopy

Fluorescence Lifetime Imaging and Fourier TransformInfrared Spectroscopy of Michelangelo’s David

DANIELA COMELLI,* GIANLUCA VALENTINI, RINALDO CUBEDDU, andLUCIA TONIOLOINFM-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 ofart based on a portable system for fluorescence imaging integratedwith analytical measurements on microsamples. The method allowsus to localize and identify organic and inorganic compounds presenton the surface of artworks. The fluorescence apparatus measuresthe temporal and spectral features of the fluorescence emission, ex-cited by ultraviolet (UV) laser pulses. The kinetic of the emission isstudied through a fluorescence lifetime imaging system, while anoptical multichannel analyzer measures the fluorescence spectra ofselected points. The chemical characterization of the compoundspresent on the artistic surfaces is then performed by means of an-alytical measurements on microsamples collected with the assistanceof the fluorescence maps. The previous concepts have been success-fully applied to study the contaminants on the surface of Michel-angelo’s David. The fluorescence analysis combined with Fouriertransform infrared (FT-IR) measurements revealed the presence ofbeeswax, which permeates most of the statue surface, and calciumoxalate deposits mainly arranged in vertical patterns and related torain washing.

Index Headings: UV fluorescence; Time-resolved spectroscopy; Im-aging; Fourier transform infrared spectroscopy; FT-IR spectros-copy; Marble sculptures.

INTRODUCTION

In the field of artwork conservation and care, nonde-structive analyses performed in situ have gained increas-ing importance in the last years because they providevaluable information in real time on the materials presenton artifacts. Such materials can be original materials usedby the artist, new formation materials deriving from de-terioration processes, and restoration materials that havebeen applied on the surfaces throughout the centuries.

Among the nondestructive investigations that havebeen applied in situ, diffuse reflectance,1–3 either in visi-ble and near-infrared spectral bands, and several spectro-scopic techniques, like optical fluorescence,1,4 X-ray fluo-rescence,5,6 and vibrational spectroscopy,6,7 are the mostattractive. Laser-induced breakdown spectroscopy hasalso been applied to the analysis of works of art; never-theless, this technique is just minimally invasive, since amicroscopic portion of the object is destroyed.3,8

Aside from the physical parameters that are specificallymeasured by any technique, a very important classifica-tion splits the investigation methods into imaging meth-ods, such as diffuse reflectance and radiographic record-ing, and non-imaging methods, such as most of the spec-troscopic measurements. The compositional heterogene-

Received 11 January 2005; accepted 9 June 2005.* Author to whom correspondence should be sent.

ity of any artwork, which is very often an important partof the author’s message, makes imaging techniques moresuitable than point measurements to understand the com-position of works of art, since they preserve the mor-phology of the object. On the other hand, point measure-ments usually provide a richer data set (e.g., a high-res-olution spectrum), while imaging methods very oftengive only an intensity map in a specific spectral band.

Fluorescence lifetime imaging (FLIM) is a techniquethat measures at once the amplitude and the decay timemaps of the fluorescence emission of a sample after ex-citation with very short pulses of ultraviolet (UV) light.FLIM has been successfully applied to several fields, in-cluding combustion analysis,9 fluorescence microscopy,10

and medical diagnosis.11,12 In the field of artwork conser-vation, it gives an immediate perception of the artworkmorphology, together with functional information. Infact, the amplitude map mainly depends on the emissionintensity and shows the artwork shape, while the lifetimemap is correlated with its compositional heterogeneity,since any compound usually exhibits a characteristic fluo-rescence lifetime.

Ultraviolet-induced fluorescence is specially suited tothe study of organic compounds, which are largely pres-ent in works of art; yet, inorganic contaminants can alsobe revealed through an indirect effect on the backgroundfluorescence emission.

Notwithstanding its effectiveness, optical fluorescencecannot provide an exhaustive characterization of a samplealone, since the emission is typically due to a mixture ofseveral chromophores. Moreover, the molecular environ-ment experienced by chromophores, which in turn mightchange with age, conservation status, etc., often influenc-es the properties of the emission.13 Thus, the extractionof analytical information from fluorescence measure-ments is not straightforward, but requires the support ofother measurements. However, FLIM is a powerful toolto distinguish between regions showing different com-pounds, which can be discriminated on the basis of theiremission lifetime.

Starting from these considerations, we developed aportable system for advanced fluorescence imaging andspectroscopy and we applied it to the analysis of surfacesof artistic interest. The system is made of two main units:a FLIM device and a spectroscopic unit based on an op-tical multichannel analyzer (OMA). The information ob-tained with fluorescence measurements indicates thepoints on the artwork at which to take microsamples tobe analyzed in the laboratory by means of techniques

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such as optical microscopy and Fourier transform infra-red (FT-IR) spectroscopy. Analytical measurements givea synergic effect with fluorescence imaging: in fact, thechemical identification of the materials provided by an-alytical techniques can be transferred to the whole exten-sion of the artwork thanks to the imaging capabilities ofthe FLIM apparatus, without the need for extensive mi-crosampling.

This measurement protocol has been previously ap-plied to assess the conservation status of renaissance fres-co paintings in Castiglione Olona.14 This experience, ma-tured through the study of mural paintings, suggested tous the application of the combination of FLIM and FT-IR techniques to investigate the contaminants on the sur-face of Michelangelo’s David within a diagnostic pro-gram aimed at supporting the conservative work carriedout in 2004. The present paper reports on the most sig-nificant results achieved in this measurement campaign.

MATERIALS AND METHODS

Fluorescence Experimental Setup. The fluorescenceimaging system used for this study is similar to the onedescribed in Cubeddu et al.11 It is based on a time-gatedintensified charge-coupled device (ICCD) (ICCD225,Photek, St Leonards-on-Sea, England) exhibiting a min-imum gate width of 5 ns. A sequence of images is ac-quired by activating the gate of the image detector atdifferent delays with respect to the excitation pulses. Inthis way, the temporal behavior of the fluorescence isrecorded in each pixel. Then, by applying a suitable fit-ting procedure, which will be described in the followingsubsection, the fluorescence lifetime map of the field ofview is reconstructed.

The UV (l 5 337 nm) excitation light is provided bya nitrogen laser (LN203C Laser Photonic, Orlando, FL)that generates 1 ns pulses. The excitation beam is coupledto a silica fiber and delivered to the sample. A homemadetrigger circuit and a precision delay generator (DG535,Stanford Research Systems, Sunnyvale, CA) allow thetemporal sampling of the emitted fluorescence. Thewhole system has been assembled in a portable rack, ex-cept the gated camera, which is connected to the controlunit through a 10 m cable for remote access to any partof the sculpture.

An optical multichannel analyzer (OMA EG&GPrinceton Applied Research, Princeton, NJ) completesthe experimental apparatus. It measures fluorescencespectra from 400 to 800 nm, with a spectral resolutionof 1 nm. A second nitrogen laser (VSL-337ND-S, LaserScience Inc.) provides the excitation light. The laserbeam is coupled to a silica fiber bundle, which is put ingentle contact with the sample through a metallic spacercovered with a Teflon ring. The bundle is made of a cen-tral fiber, which delivers the excitation light to an area 3mm in diameter, and 20 fibers arranged in two circularrings, which collect the emitted fluorescence. The spacermaintains the fibers at a suitable distance from the samplein order to optimize the superposition of the excited areawith the field of view of the collection fibers.

Data Analysis. Fluorescent systems can hardly bemodeled as mono-exponential due to the simultaneouspresence of several emitting molecules in the same spec-

imen. Nevertheless, even in the case of multi-exponentialbehavior of the fluorescence emission, a linear fit per-formed on the logarithm of acquired data allows the re-construction of an effective lifetime.15 The lifetime mapyields strong contrast for the discrimination of differentcompounds, as has also been shown by other researchgroups.16 Further, the processing of a high-resolution im-age with a linear fitting algorithm requires only a fewseconds. Therefore, such algorithms would also be suit-able for real-time data processing.

Assuming a mono-exponential behavior of the fluores-cent emission f , with effective lifetime t and ampli-tude A:

f (t) 5 A exp(2t/t) (1)

the fluence H acquired in each pixel as a function of thedelay d, is given by the following formula:

d1w

H(d) 5 C f (t) dtEd

d d 1 w5 CAt exp 2 2 exp 21 2 1 2[ ]t t

w d5 CAt 1 2 exp 2 exp 2 (2)1 2 1 2[ ]t t

where w is the gate width and C is a constant dependenton the efficiency of the detection system. The effectivelifetime t and the amplitude A can be reconstructed by aleast mean squares fit performed on N time samples, lead-ing to the following equations:

2

2N d 2 dO Ok k1 2k k

t 5 2 (3)N d ln H(d ) 2 d ln H(d )O O Ok k k k

k k k

21H(d ) d wk kA 5 H(d ) exp 1 2 exp 2 (4)O k 1 2 1 2[ ]t t tk

Both t and A are matrices that represent the spatial mapsof the fluorescence lifetime and amplitude of the samplein the field of view of the gated camera.

As far as the meaning of the two matrices t(x, y) andA(x, y), the first reveals areas with different chemicalcomposition, while the second gives information on therelative abundance of the fluorescent materials in the fieldof view. By merging the two maps, a third one, namedthe HSV map, is created. This map is based on the HSV(Hue, Saturation, and Value) color model.17 The lumi-nance (value) of each pixel is proportional to the fluo-rescence amplitude, while the hue represents the lifetime,keeping the saturation constant at 0.8. In this way, theHSV map allows one to easily associate the functionalinformation provided by the lifetime (hue) to the mor-phology of the analyzed region, given by the fluorescenceamplitude (value).

Experimental Procedure. For the measurements onDavid, the gate of the image detector was set to 100 ns,wide enough to get almost all the fluorescent light emit-ted by the sculpture. A set of 12 images was recorded,corresponding to delays of 0, 2, 3, 5, 8, 10, 12, 15, 20,

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

30, 40, and 50 ns with respect to excitation pulses. Then,the images were processed in order to calculate the FLIMmaps. From the reconstructed maps, it was possible todistinguish regions showing similar characteristics, aswill be described hereafter. In correspondence with eachhomogeneous region, a fluorescence spectrum was re-corded and, at a few selected points, a microsample ofthe material superimposed on the sculpture was takenwith a scalpel and the aid of a portable microscope; onlymaterials overlaying the marble surface were sampled; nosampling of the sculpture itself was allowed.

Microsamples were examined with a Fourier transforminfrared spectrophotometer (Nicolet Nexus) coupled to anFT-IR microscope (Nicolet Continuum). The detector wasa liquid N2 cooled HgCdTe detector. Spectra were re-corded using a Graseby–Specac diamond cell accessoryin transmission mode between 4000 and 700 cm21.

RESULTS AND DISCUSSION

An extensive measurement campaign was carried outover the David’s entire surface. A total of 45 fluorescenceimages were collected in areas representative of conser-vation status and geometrical orientation, which could berelevant for the accumulation of deposits or for the actionof atmospheric agents. To this purpose, we consideredareas with different surface roughness or with differentslope with respect to the vertical direction. Also, the vi-sual inspection and a set of photographs taken with aWood lamp indicated several areas of interest, e.g., areasshowing a yellowish color in white light or bright spotsin UV light.

A common finding that was observed in all the areasconsidered in this study was the unexpected intense emis-sion coming from the David’s surface in response to UVexcitation light. It is important to emphasize that miner-als, excited by UV light, often emit a characteristic lu-minescence; the emission is due to impurities located in-side the crystal lattice of the material. In marble, for ex-ample, Mn21 can substitute for Ca21 ions, becoming themain emitting centers, whereas Sm31 and Dy31 are minordefects. Yet, studies on calcite, of which marble is made,have shown that these types of impurities are character-ized by a luminescence having a decay time on the orderof microseconds,18,19 thus non-detectable with our fluo-rescence setup, which works on the nanosecond timescale. Furthermore, Carrara marble has a very low con-centration of Mn21 ions.

To confirm the low fluorescence expected from calci-um carbonate, a quarry sample of Carrara marble wasmeasured in our laboratory with the same experimentalsetup used to analyze the Michelangelo masterpiece. Theemission from the quarry sample was on average 2–3times lower than that from the David and showed a peakaround 500 nm, while the peak of the David’s emissionis always beyond 500 nm and sometimes is even beyond550 nm.

Figure 1 shows three normalized spectra that well-de-pict this concept: spectrum a refers to the sample of quar-ry Carrara marble, spectrum b was taken from the Da-vid’s surface close to the right ear lobe, and spectrum cwas measured from a small fragment of the David, a fewmillimeters thick, collected from the second toe of the

left foot after a vandalism carried out in 1991. This lastspectrum is especially interesting since it was taken fromthe inner surface of the fragment. The differences be-tween spectrum a and spectra b and c are evident, mainlyconcerning the peak position. Also, the fluorescence life-time easily distinguishes the Carrara quarry sample fromthe David’s marble. In fact, even if the time behavior ofboth emissions can hardly be modeled by a mono-expo-nential decay, the effective lifetime of Carrara marblefluorescence is definitely longer (.7–8 ns) than that ofthe David’s marble by at least 2 ns.

Hence, the differences in spectral features, temporalbehavior, and intensity between the fluorescence of Car-rara marble and that of David’s marble let us supposethat David’s surface is extensively permeated by organicmaterials, which have their own emission properties anddetermine the fluorescence features of the statue muchmore than the impurities, naturally occurring in the mar-ble microstructure, can do. This observation can be ex-plained as the result of centuries of environmental con-tamination and as the consequence of several conserva-tion treatments carried out in the past.

In the following, some details exemplificative of theorganic materials and inorganic salts more frequentlyfound on the David’s surface will be discussed.

Some small fluorescent spots, sporadically present onthe surface, e.g., on the right forearm, on the back, onthe shoulders, and on the trunk, have a bright emissionand appear light blue under the Wood lamp. They havea fluorescence lifetime greater than 6 ns (Fig. 2) and aspectral peak around 520 nm. Such spots are well out-lined both in the amplitude and in the lifetime maps andwere identified as composed of beeswax through the FT-IR analysis20 (Fig. 3 and Table I) of a microsample col-lected in the back of the statue, on the left of the sling.

Since the fluorescence decay time of the drops of bees-wax is the longest ever found on the statue, using thelifetime maps collected all over the marble surface, it waspossible to localize most of such wax remains. The pres-ence of large quantities of wax in the small dips thatcharacterize the marble finishing of the sling, located onthe back of the statue (data not shown), confirms that the

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FIG. 2. Fluorescence analysis performed on the right forearm of theDavid: (a) color picture of the area; (b) fluorescence lifetime map; and(c) fluorescence amplitude map. The maps show the presence of fluo-rescent spots on the marble surface characterized by a lifetime close to6 ns.

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

TABLE I. Infrared frequencies of minerals and organic com-pounds of the collected spectra.

Compound/mineralFrequency

(cm21) Vibration

Beeswaxa 29552918285017421468137911751100

722

stretch. nas C–Hstretch. nas CH2

stretch. ns CH2

stretch. ns C5Osciss. ds CH2

bend. sym. CH3

wag. CH2

stretch. ns C–Crock. CH2

Whewellite 1631 stretch. nas CO22

(calcium oxalate monohy-drate)a,b

1316 stretch. ns CO22

780Gypsum 3543 stretch. n –OH (H2O)

(calcium sulfate dihy-drate)b

3407168316211128

672

stretch. n –OH (H2O)bend. H–O–Hbend. H–O–Hstretch. SO4

22

Calcite 1798 stretch. nas CO322

(calcium carbonate)b 1442875713

stretch. ns CO322

Quartz 1080 stretch. nas Si–O(silicium dioxide)b 798

780

a Ref. 21.b Ref. 22.

whole David’s surface underwent a conservation treat-ment based on beeswax in 1813, as can be inferred fromarchive documents.23 Such treatment, called ‘‘encausto’’,was carried out to protect the statue from rainfall andother atmospheric precipitations. According to the recipereported in ancient treatises, the encausto was based onhot wax; after about 200 years, not withstanding severalcleanings, wax is still widely present on the statue, asevidenced by FLIM measurements combined with FT-IRspectroscopy.

As a further example of these findings, Fig. 4 showsthe lifetime (Fig. 4b) and the amplitude (Fig. 4c) of theback of the David’s left hand. The lifetime map is ratheruniform, while the amplitude map presents a pattern thatis reminiscent of the typical marble veins, also visible inthe white-light image (Fig. 4a). Actually, the amplitudemap accounts for absorbance differences due to the pres-ence of metal cations that give the marble its character-

istic gray color and simply act as absorbers for the ex-citation light. On the contrary, the lifetime only dependson the chemical structure of the fluorophores uniformlyadsorbed into the crystalline structure. Figure 5 showsthe emission spectrum of the left hand compared with thespectrum taken in one of the fluorescent spots shown inFig. 3. The lifetime (6 ns) and the spectrum of the emis-sion taken in the left hand are similar to those of wax,confirming that the material distributed all over the sculp-ture is very likely based on this organic compound.

Wide regions of the statue present a vertical patternmade of irregular stripes that can hardly be perceived in

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

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 pos-sible to observe a vertical stripe characterized by a short lifetime value,close to 4 ns. Very likely, in this region the fluorescence emission ofthe marble, mostly due to the underlying wax residues, is dumped bythe presence of inorganic salts and metal cations.

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 theright forearm. The similarity of the emissions suggests that the twosurfaces are permeated by the same compound.

white light and that give a dark brown emission on in-spection under the Wood’s lamp. The fluorescence mapsof these details present low amplitude and short lifetime(around 4–5 ns), whereas those parts of the surface thatappear clean and healthy have a longer lifetime ($6 ns),similar to the one measured in the left hand. Figure 6shows an example of such a region, located in the rearpart of the left thigh. Upon visual inspection, the mor-phology of the short lifetime sectors can be correlated tosurface erosion where salts and particulate matter are pre-sent. The FT-IR spectrum (Fig. 7 and Table I) of thebrownish deposits established that they generally containgypsum (calcium sulfate dihydrate), along with weddeliteor whewellite (calcium oxalate dihydrate or monohy-drate), calcite (calcium carbonate), and quartz (silicon di-oxide).

APPLIED SPECTROSCOPY 1179

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

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

This surface alteration should be ascribed to the out-door exposition of David sculpture until 1873, when thestatue was placed inside the museum ‘‘Galleria dell’ Ac-cademia’’. Deposits or surface patinas of this type (acompact mixture of different minerals often containingsmall amounts of organic compounds) have been formedover the centuries.23,24 A complete characterization of thepatina could not be achieved since the amount of materialwe were allowed to take from the statue was just enoughto perform vibrational spectroscopy, while gas chroma-tography and mass spectrometry, which are more suitedto the study of the organic fraction of the patina, wereprecluded. Nevertheless, some insight can be gained fromfluorescence images. In fact, in correspondence with thepatina, the fluorescence of the marble, mostly due to theunderlying wax residues, is decreased to shorter lifetimeand lower amplitude. While the decrease in amplitude canbe easily explained by the shielding effect of the patinain the outermost surface, the change in lifetime can leadto different interpretations. It can possibly be ascribedeither to the presence in the patina of a low amount offluorescent compounds with a lifetime shorter than thatof the beeswax or to some kind of interaction betweenthe beeswax and the inorganic salts of the patina, leadingto an increase in relaxation pathways of the excited states.

Finally, Fig. 8 shows the HSV map of the lower partof David’s face. A small red spot under the right nostrilpresents the typical lifetime of wax residues (.6 ns),while the larger yellow spot above the lips has a slightlyshorter lifetime (5.8 ns). The two contaminants alsogreatly differ in spectral features (Fig. 8c). In fact, thespectrum of the spot under the nostril corresponds to thatof beeswax, as expected from its lifetime, while the spec-trum of the spot over the lip peaks at a definitely longerwavelength. The appearance of this spot led us to supposethat it should be made of organic material, but it was notpossible to assess its chemical structure, since microsam-pling from the David’s face was not permitted.

The analysis of many other details of the David’s sur-face allow us to conclude that three main types of over-laid materials were largely mapped by fluorescence im-

aging: (1) beeswax residues concentrated in small dropsor permeated into the marble surface; (2) salt deposits,mainly composed of gypsum, calcium oxalates, and par-ticulate matter; and (3) some organic contaminants, notprecisely identified, located in small areas.

The FLIM apparatus, along with other devices, wasalso tested to compare different cleaning methods appliedto small test areas on the statue. As an example, cleaningtests were performed in a region located on the left shin,characterized by the presence of inorganic deposits(mainly composed of gypsum). Figure 9a shows twopatches that were treated with different cleaning proce-dures: the upper patch (G1) was cleaned with a deionizedwater poultice, while the lower patch (G2) was cleanedwith ion exchange resin (DES90). The fluorescence life-time maps of the two areas taken before (Fig. 9b) andafter (Fig. 9c) the cleaning are also shown.

The increase in the fluorescence lifetime that takesplace after the cleaning (red shift of the false color map)

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

in both patches indicates that some inorganic depositshave actually been removed by the cleaning procedures.In fact, the reduction in fluorescence dumping associatedwith the very superficial layer let the long-lived emissionof beeswax absorbed in the marble microstructure be-come more relevant for the calculation of the effectivelifetime. Moreover, the slightly greater increase in thelifetime shown by the G1 patch indicates that the waterpoultice is possibly more effective than ion exchange res-in.

CONCLUSION

The analysis of David by FLIM was a challenging taskfor our research unit. Michelangelo’s masterpiece is as-tonishing for its size: it is more than 5 m tall and itssurface area is about 20 m2. Such a large area can beconveniently mapped only with a wide-field imaging sys-tem. On the other hand, the field of view of our FLIMdevice is very often restricted to a few tens of centimetersby the emission intensity of the sample, which is excitedby a low-power laser source. Marble is made of calcitegrains that, being constituted of CaCO3, should not fluo-resce when irradiated with UV light at 337 nm. Yet im-purities always present in marble, beyond a well-knownluminescence, also give it a faint fluorescence peaking at500 nm, as we found with our sample of Carrara marble.If the David fluorescence was to be ascribed only to thebulk material, such a faint emission could have beenhardly detected with our system. Unexpectedly, the fluo-rescence signal was strong enough to allow us to takewide images of the statue from a distance of 0.5–1 m,which is the minimum required to approach the Davidunder safe conditions and for a reasonable mapping ofsuch a wide surface. This result has been interpreted as-suming that a large amount of organic material is ab-sorbed into the marble. This finding is possibly a generalcondition of all ancient marble sculptures having a longhistory of outdoor exposition and conservation practice.Our experience with another sculpture of Michelangelo’s(Pieta Rondanini, hosted in the museum ‘‘Civiche Rac-colte del Castello Sforzesco’’ in Milan) seems to confirmthis hypothesis. The widespread presence of organic con-taminants on marble sculptures gives our technique agreat relevance among the noninvasive diagnostic pro-cedures. The measurement campaign with David showedthat the synergic combination of FLIM with spectroscopyand other laboratory measurements on microsamples(such as FT-IR examination) allowed us to identify someof the overlapped materials. This is especially true forbeeswax, which has been found all over the statue as abackground signal and in well-outlined spots, sometimeslooking like drops. Also, most of the inorganic depositswere mapped thanks to the decrease in the lifetime of theunderlying fluorescence emission.

Fluorescence lifetime imaging has already been suc-cessfully applied to the analysis of fresco paintings,14

while the investigation of oil paintings and other paint-ings is in progress. The results achieved up to now let ussuppose that a well-designed investigation protocol basedon FLIM, combined with analytical techniques, could beprofitably 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’ Acca-demia, Dr. Franca Falletti, and the scientific team coordinator, Dr. MauroMatteini, for their valuable and constant support during the measure-ments and the analysis of data. Particular thanks is due to Dr. A. Ald-rovandi for the valuable collaboration and the availability of the pho-tographic materials.

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24. L. Rampazzi, A. Andreotti, I. Bonaduce, M. P. Colombini, C. Co-lombo, and L. Toniolo, Talanta 63, 967 (2004).