Journal of Cultural Heritage 61 (2023) 91–99

Contents lists available at ScienceDirect

Journal of Cultural Heritage

journal homepage: www.elsevier.com/locate/culher

VSI: LACONA XIII

Insights into the stratigraphy and palette of a painting by Pietro

Lorenzetti through non-invasive methods
Alice Dal Fovo a,∗, Sara Mattana a, Alessandra Ramat b, Patrizia Riitano b, Riccardo Cicchi a,c,
Raffaella Fontana a
a
National Institute of Optics - National Research Council (CNR-INO), Largo E. Fermi 6, 50125, Florence, Italy
b
Opificio delle Pietre Dure, Viale F. Strozzi 1, 50129, Firenze, Italy
c
European Laboratory for Non-Linear Spectroscopy (LENS), Via Nello Carrara 1, 50019, Sesto Fiorentino, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Paintings are complex multi-layered structures characterised by high compositional and structural het-
Received 3 January 2023 erogeneity. Therefore, a combined analytical approach is often required to overcome ambiguities in the
Accepted 7 March 2023
interpretation of data acquired by an individual technique. In this paper, we applied three non-invasive
imaging techniques for the study of a 14th-century altarpiece by Pietro Lorenzetti belonging to the Uffizi
Keywords: Galleries. In detail, we used reflectance imaging spectroscopy to identify and map the pigments, and re-
Pigment mapping veal features not visible to the naked eye; to quantify the colorimetric differences induced by the clean-
Lorenzetti ing; to visualise and measure the micrometric thickness of the paint layers. Then, we applied optical
Reflectance imaging coherence tomography to visualise and measure the micrometric thickness of the material layers. Finally,
Optical coherence tomography
we carried out fluorescence lifetime imaging to discriminate the pictorial integration from the original
Fluorescence lifetime imaging
painting using a method never before applied to a work of art. The data was discussed within a multi-
disciplinary team of experts.
© 2023 Consiglio Nazionale delle Ricerche (CNR). Published by Elsevier Masson SAS. All rights reserved.

1. Introduction
Paintings are complex multi-layered systems characterised by
extremely varied chemical compositions and physical properties.
Due to the fragility and value of such objects, it is preferable -
whenever possible - that the analyses are carried out in situ, with-
out sampling and contact between the instrument and the object.
For several decades, the non-invasive multi-technique approach
has proven effective in providing morphological, structural and
chemical information on a wide range of artworks [1,2]. Numerous
optical techniques are now jointly applied to overcome ambiguities
in the interpretation of data acquired by the individual techniques,
yielding information on a micro- and macro-metric scale without
causing any damage to the artwork. For instance, pigments iden-
tification can be achieved by combining elemental and/or molecu-
lar analysis with Reflectance Imaging Spectroscopy (RIS) [3], which
is based on the detection of characteristic spectral features deter-
mined by electronic or vibrational transitions and detected in the
Vis-IR spectral range. Multivariate Statistical (MVS) methods [4,5]
∗
Corresponding author.
E-mail address: alice.dalfovo@ino.cnr.it (A. Dal Fovo).
are typically used for reducing the dimensionality of RIS data and
for the identification of characteristic reflectance spectra in the im-
age cube. Examination of spectral features integrated with infor-
mation obtained by other non-invasive mapping methods, such as
X-Ray Fluorescence (XRF) imaging [6], allows for the identification
of pure pigments, mixtures of pigments or stratifications. The lat-
ter are used to create labelled maps of pigment distribution over
the painting surface. Spectral angle mapping (SAM) [7] and Spec-
tral Correlation Mapping (SCM) [8] are among the most widely
used MVS methods for pigment mapping. The main issue with
these approaches is that they consider the spectrum as a linear
combination of two or more endmembers, which ideally represent
the pure pigments, and thus are based on linear spectral unmixing
models. However, the reflectance from mixtures of pigments com-
monly found in paintings gives a nonlinear response as a result
of the scattering between either the pigments mixed in different
proportions or applied as superimposing layer [9]. Recent studies
show that some alternative approaches can overcome the limita-
tions of linear unmixing. One possible solution can be the use of
a simplified Kubelka-Munk (KM) model for opaque and infinitely
thick samples, which approximates the reflectance from mixed pig-
ments, at the expense of intensive computational operations [10].
Deep Learning (DL), especially Neural Networks (NN), has been

https://doi.org/10.1016/j.culher.2023.03.002
1296-2074/© 2023 Consiglio Nazionale delle Ricerche (CNR). Published by Elsevier Masson SAS. All rights reserved.
A. Dal Fovo, S. Mattana, A. Ramat et al.
recently introduced for the a priori pigment selection in hyperspec-
tral data cubes, showing promising results [10]. However, to create
accurate material maps with NN, huge training datasets of labelled
reflectance spectra must be generated [11].
One of the most applied methods for stratigraphic non-invasive
analysis is Optical Coherence Tomography [12], an interferomet-
ric technique providing 2D and 3D images of the internal struc-
ture of paint layers with micrometric resolution. Many applications
have demonstrated the efficiency of OCT in the survey of semi-
transparent material layers. However, opaque or highly scattering
materials, such as many pigments and varnish, may obstruct the
radiation and prevent in-depth visualisation of the painting stratig-
raphy [13].
The presence of intrinsically luminescent materials can be eval-
uated with UV-excited fluorescence, which has long been applied
as a simple photographic technique for the non-invasive qualita-
tive examination of artworks [14]. More detailed analysis of or-
ganic and inorganic compounds, including proteins, oils, waxes
and resins can be performed by spectrally resolved laser-induced
fluorescence (LIF) [15]. However, several factors can strongly af-
fect the overall emissions. Among the others, intrinsic similari-
ties in the emission spectra of different fluorophores, the influ-
ence of the molecular environment, as well as the presence of
non-fluorescent pigments dispersed in protein- and oil-based or-
ganic binders, makes the interpretation of fluorescence signal ex-
tremely complex. It has been demonstrated that the analysis can
be greatly facilitated by resolving the emission decay dynamics of
the fluorescence emission. Specifically, Fluorescence Lifetime Imag-
ing (FLI) [16] is particularly well suited for spatial mapping of com-
positional heterogeneities over an examined area, being insensitive
to changes in the concentration of the emitting material, as well as
being independent of the fluorescence intensity [17].
In this work, we used a non-invasive, multi-analytical approach
for the identification and mapping of pigments and the study of
the micro-stratigraphy in the Saint Humility altarpiece by Pietro
Lorenzetti (Uffizi Galleries, Florence). Two of the panels of the al-
tarpiece were examined using three imaging techniques, namely
multispectral RIS in the Vis-NIR, OCT, and FLI. A novel portable
fibre-based system was used to discriminate organic and inorganic
luminescent mixtures based on their different fluorescence life-
times. In our recent preliminary works [18,19], we propose the use
of a novel handheld fibre-based FL system [20] to discriminate lu-
minescent organic and inorganic compounds, such as natural resin,
oil and wax [18] and to study the influence of the pigment dis-
persed in the binder on the fluorescence signal detected on paint
samples [19].
RIS analysis here reported relies on previous results obtained
with SCM and macro-XRF imaging on Panel 1 before the clean-
ing process [21]. On that occasion, the FL setup was applied for
the first time on artwork to explore the possibility to relate the
phasor of the reference samples (i.e. pigment dispersed in the egg
binder) to the composition of the paint mixtures on Lorenzetti’s
painting. SCM and FL mapping were carried out, based on a set of
egg tempera samples, specifically prepared according to the histor-
ical recipes. Here, pigment identification and mapping were car-
ried out with RIS on both panels after the cleaning phase, using
the same reference samples. Imaging in the near-infrared range re-
vealed hidden details, not visible to the naked eye. Colour differ-
ences before and after the cleaning of Panel 1 were identified by
Principal Components Analysis (PCA) and quantified by colorimet-
ric analysis. The stratigraphy of areas of interest, i.e. overpainting
and in-painted areas, was studied by OCT. Finally, the same re-
gions were mapped with FLI, through the application of the phasor
method and the Gaussian Mixture Model (GMM) to explore the po-
tential of the technique to identify different emitting species in an
unsupervised and automated manner.
92
Journal of Cultural Heritage 61 (2023) 91–99
1.1. Research aim
The aims of this study were the following: to identify and map
the main pigments used by Lorenzetti for two of the panels per-
taining to the Saint Humility altarpiece; to reveal details under-
neath the painted surface that are not visible to the naked eye;
to quantify the colorimetric differences induced by the removal of
foreign material through the cleaning operation; to visualise and
measure the micrometric thickness of the paint layers; to discrim-
inate the in-painting from the original areas using a method never
before applied on a painted artwork.
2. Materials
2.1. Case study
As a case study, we analysed two panels of the altarpiece “Saint
Humility and stories of her life” (257 × 168 cm), tempera on wood,
gold background, painted by the Sienese artist Pietro Lorenzetti be-
tween 1335 and 1340, now in the Uffizi Galleries (Inventory 1890
nos. 6120–6126, 6129–6131, 8347) [22]. The altarpiece is composed
of the central full-length image of Saint Humility (1226–1310) sur-
rounded by panels that recount key moments of her life and mir-
acles. We analysed two panels depicting the story of a Vallom-
brosian monk that refuses to have a sick foot amputated (from now
on identified as Panel 1), and Saint Humility’s escape from the con-
vent and crossing of the river Lamone (Panel 2), as seen in Fig. 1a,
b. The episodes are set within architectural or landscape views and
abound in details derived from the daily life of the time.
The altarpiece was in the church of San Giovanni Evangelista,
Florence, which was founded by Saint Humility herself in 1282. In
1529 ca., the nuns had to leave the convent due to the siege of
Florence and the painting was then removed from its original lo-
cation. Over the centuries, the altarpiece has been dismembered
and is now without a frame: two pinnacles and two of the story
panels, are now in Berlin (Staatliche Museen, Gemäldegalerie). The
panels in the Uffizi Galleries are currently under restoration at the
Opificio delle Pietre Dure.
2.2. Reference samples
A series of egg tempera paintings on wooden support was used
as a reference for the identification of the artist’s palette. All sam-
ples were prepared by the Opificio delle Pietre Dure according to
the late Medieval and Renaissance recipes. Pure powdered pig-
ments (by ZecchiTM , Florence), whose chemical composition was
previously verified by FT-IR, were dispersed in a proteinaceous
binder (2/3 yolk, 1/3 egg white, 1/3 white vinegar) to form the egg
tempera. The paint layers were applied on a preparatory ground
made of gypsum and natural glue and finished with a rabbit glue
primer.
3. Instruments and methods
3.1. Reflectance imaging spectroscopy (RIS)
The multispectral scanner, developed at the National Research
Council-National Institute of Optics (CNR-INO), acquires simultane-
ously 32 narrow-band images (16 VIS + 16 NIR) comprising point-
wise spectral information in the range 395–2550 nm. A detailed
description of the device can be found in the literature [23]. The
painting surface is illuminated through whiskbroom scanning by
two low-voltage current-stabilized halogen lamps and two narrow-
spot high-power white LEDs. A square-shaped fibre bundle collects
the back-scattered light from a single point of the scanned sur-
face and delivers it to a set of Si and InGaAs photodiodes, each of
A. Dal Fovo, S. Mattana, A. Ramat et al.
Fig. 1. RGB image of two panels from the altarpiece by Pietro Lorenzetti of the Uffizi Galleries (Florence), (a) “A Vallombrosian monk refuses the amputation of his infected
foot” (Panel 1 – after cleaning) and (b) “Saint Humility escapes from the convent and crosses the river Lamone” (Panel 2).
them equipped with an interferential filter. The optical head, com-
posed of the lighting system and the collecting optics, is placed in
a 45°/0° illumination/detection geometry and is moved by an XY
scanning system with a 250 μm step (4 points/mm) and 500 mm/s
speed, resulting in 3 h acquisition time for the maximum scan-
ning area of 1 m2 . A proper calibration procedure was performed
by measuring a certified white 100% reflectance reference stan-
dard (SpectralonTM ) and background noise, following CIE indica-
tions for non-contact spectrophotometric measurements. The opti-
mal target-lens distance is ensured during the scanning by an aut-
ofocus system based on a high-speed triangulation distance metre.
The output is a stack of perfectly superimposing monochromatic
images, metrically correct and free from aberrations.
3.1.1. SCM, PCA
The monochromatic images were processed with software de-
veloped in-house and specifically tailored to the analysis. Spectral
mapping was performed with an SCM algorithm [24], which con-
siders spectra as vectors in N-dimensional space, where N corre-
sponds to the number of spectral bands. The angle between the
reference (R) and target spectra measures their similarity – i.e.
the smaller the angle, expressed in radians < 0, π >, the higher
the degree of similarity. In SCM images, the pixel intensity is pro-
portional to the similarity between the vector representing the
spectrum of each pixel and the reference (or endmember) being
mapped. A close match means a high-intensity value in the image
plot. SCM represents an improvement of SAM, as it centralises the
data in its mean considering also the negative correlation. SCM re-
lies on the calculation of similarity (−1 < R < 1), where 1 means
total correlation, through Pearson’s correlation coefficient, yielding
more accurate classification results. All SCM maps were obtained
by selecting a spectral similarity range between 0.95 and 1. Con-
sequently, the areas highlighted in the maps (pixels with high in-
tensity) indicate with good confidence the presence of the pigment
used as a reference.
RIS data cubes of Panel 1 were also analysed with PCA be-
fore and after the cleaning. This unsupervised exploratory method
looks for directions of maximum variance within a multivariate
data space, where high variance (i.e., high variability) means a
large amount of information. The directions of variance (PCs) are
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Journal of Cultural Heritage 61 (2023) 91–99
orthogonal and thus uncorrelated, i.e. the information contained is
never redundant [25]. The coefficients that multiply each variable,
called loadings, represent the cosine values of the angles between
the PCs and the original variables. We compared the loading val-
ues of the original variables on three selected PCs, providing infor-
mation about the importance of the variables and their intercor-
relation, with respect to the fraction of variance (i.e., information)
explained by those PCs. Colour composite images were also elab-
orated by combining three PCs in the trichromatic RGB space and
PC data were processed in Matlab environment.
3.2. Spectral-domain optical coherence tomography (Sd-OCT)
The OCT device used in this study is a Thorlabs Telesto-II, which
incorporates a superluminescent diode (1300 nm central wave-
length and about 170 nm bandwidth) with an axial resolution of
5.5 μm in air and lateral resolution of 13 μm. The maximum field
of view (FOV) is 10 × 10 mm2 , with a 3.5 mm imaging depth. The
detection system consists of a spectrograph (a diffraction grating)
and a fast camera. The depth information is acquired using a Fast
Fourier Transformation (FFT). The system is controlled via 64-bit
software running on a high-performance computer. The 3D scan-
ning probe with an integrated video camera allows for high-speed
imaging (76 kHz) for rapid volume acquisition and live display. The
sample stage provides XY translation and rotation of the sample
along with the axial travel of the probe.
3.3. Fluorescence lifetime imaging (FLI)
The FLI prototype shown in Fig. SM1 in the Supplementary Ma-
terial was developed by CNR-INO. A detailed description of the
setup can be found in [20]. The excitation source is a picosec-
ond pulsed laser diode at 375 nm (BDL-SMN-375, Becker & Hickl
GmbH, Berlin, Germany) operating at 20 MHz. A 660 nm guid-
ing beam from a fibred light emitting diode (LED, M660FP1, Thor-
labs, Newton, NJ, USA) is delivered with the excitation beam to the
sample surface by two 200 μm fibres (0.22 NA). The fibre bun-
dle is handheld and can be freely moved over the sample surface
at a distance ranging from 2 to 10 mm, resulting in an irradiated
spot diameter of 0.9–4.5 mm. The fluorescence emitted from the
A. Dal Fovo, S. Mattana, A. Ramat et al.
specimen is collected by a third 200 μm fibre and spectrally nar-
rowed by an emission filter (FF01–510/84–25, Semrock, Rochester,
New York, NY, USA) centred around 510 nm (± 42 nm). The signal
is delivered to the detector (HPM-100–40, Becker & Hickl GmbH),
which is connected to a Time-Correlated Single Photon Counting
(TCSPC) acquisition card (SPC-730, Becker & Hickl GmbH). For all
measurements, the average power on the sample surface was kept
below 10 μW. Real-time fluorescence lifetime maps are acquired
at a macroscopic scale and, remarkably, under bright illumination
to allow visual control of the examined area during measurements,
as described in [18].
3.3.1. Gaussian mixture model (GMM) analysis
Fluorescence measurements were time-resolved through time-
correlated single-photon counting (TCSPC), given its higher tempo-
ral resolution, sensitivity, and dynamic range compared to other
time-resolved methods. Fluorescence intensity decay measured on
the samples’ surfaces was analysed using the phasor method, fol-
lowing our previous studies on painted mock-ups and metal-based,
varnished samples [8,19]. A comprehensive description of this ap-
proach can be found elsewhere [26]. The real and imaginary com-
ponents, g and s, of each decay curve were obtained through the
Fourier transform, from which the phase (τ -phase) lifetime was
calculated following the expression τ phase = s/(2π fg), where f was
taken as equal to the repetition rate of the laser (20 MHz). The
coordinates g and s were plotted as a single point (phasor) in
the phasor plot, a semicircle curve, also called a “universal trajec-
tory”. FL data can be analysed and interpreted considering that the
universal semicircle is a lifetime ruler for single-lifetime species,
and indicates relative changes in the lifetime for complex species
[26]. Further processing of FL data was performed with the Gaus-
sian mixture model (GMM), a clustering method that assumes all
data points as a linear combination of a finite number of Gaussian
distributions. A full description of this method is provided else-
where [27]. In brief, GMM consists of a weighted sum of com-
ponent Gaussian densities with the number of terms K being the
number of clusters, as reported in Eq. (1):

K
p( x ) = f i N ( u i σi ) (1)
i=1
where fi is the fraction of each cluster, N (ui i ) is the multivari-
ate normal distribution with two parameters characteristic of the
cluster data, ui and σi , are the mean coordinates and covariance
matrix, respectively.
This method addressed the need for the unsupervised and au-
tomatic identification of fluorescence species within a sample. To
the best of our knowledge, this is the first time GMM is employed
to analyse the fluorescence lifetime data obtained from a historical
painting.
This model was implemented with the fitgmdist Matlab func-
tion, which uses the expectation-maximization (EM) algorithm to
iteratively optimise the model until convergence is obtained: at
each iteration, the likelihood belonging to each cluster, i.e., the
expectation, is obtained by evaluating the data points under the
current model; the likelihood is then used to calculate the mean
position and the covariance matrix of every cluster, weighting the
points toward the subsequent maximisation. In this analysis, we
assumed K = 2 or 3 in each measured dataset.
4. Results
4.1. Reflectance imaging spectroscopy (RIS)
The most spectrally diverse areas were selected on both pan-
els for pigment identification. The prevalent spectral response was
94
Journal of Cultural Heritage 61 (2023) 91–99
assessed by comparing at least ten spectra acquired over the tar-
geted regions. We used the selected spectra (internal endmembers)
to compute preliminary unlabelled maps of the painted surface.
Areas identified by individual maps (not shown) were interpreted
as indicative of the use of pure pigments, whereas overlapping ar-
eas identified by multiple spectral correlated (SC) maps were at-
tributed to the presence of pigment mixtures or layering, their
colour resulting by linear addition of the combined, individual
maps. The internal endmembers were compared with the reference
spectra showing similar spectral features, to facilitate the identifi-
cation of the pure pigments composing the mixtures (Fig. SM2 in
the Supplementary Material). The spectral analysis allowed for the
identification of eleven pure pigments, which were used as exter-
nal endmembers for labelled SC mapping (Fig. 2c). The so-obtained
maps (Fig. SM2) are shown as single-colour images, where the
same colour of the relative endmember spectrum is assigned to
the intensity scale of the image. For each pixel of the SC maps, the
maximum intensity indicates the maximum spectral similarity be-
tween the reference and the painted surface, whereas black areas
mean no correlation. For each panel, all SC maps are combined in a
single image (Fig. 2a, b) evidencing the distribution of each identi-
fied pigment all over the painting. No differences were found with
respect to the previous SCM analysis integrated with XRF results
[21] performed on Panel 1 before the cleaning. Results suggest that
both Panels are painted with the same pigments palette, except for
indigo (indigotin), which was used only in Panel 1 for the blanket
on the bed. The other pigments, mainly used in a mixture, are the
following: red ochre and raw sienna (both iron oxide pigments),
orpiment (arsenic sulphide), green earth (hydrous iron and mag-
nesium alumino-silicate clay micas), natural umber (iron and man-
ganese oxide), bone black (calcium phosphate and calcium carbon-
ate), malachite and azurite (basic copper carbonates), lead white
(basic lead carbonate), and cinnabar (mercury sulphide). The latter
pigment is hardly detectable by RIS in the skin tone, but its pres-
ence is confirmed by the detection of the Hg signal by XRF [21].
The distribution of the main pigment mixture or layering all over
the painting surface of each examined panel is reported region by
region in Table 1.
Further analysis of the RIS data cube of Panel 1 revealed the
presence of hidden details not visible to the naked eye (Fig. 3).
Specifically, the extraction of the NIR image at 1292 nm allowed
visualizing the extent of the golden leaf below the top of tree fo-
liage (area 1); a possible pentimento in the architecture suggesting
that some windows were arranged differently in the underdrawing
(area 2); in-painting of missing parts, e.g. the feet of the figure on
the right (area 3).
PCA was performed on the RIS data cubes acquired before and
after cleaning of Panel 1. The first three PCs, which explain more
than 98% of the overall variability, were used to obtain the colour
composite images in the trichromatic RGB space (Fig. 4a, b). The
loading values of the three selected PCs were plotted as a function
of wavelength in the spectral range of acquisition (Fig. 4c, d). The
plots highlight the intercorrelation of the variables with respect to
the fraction of variance (i.e., information) explained by the PCs be-
fore and after the cleaning. Visual comparison of the RGB image
composites allowed selecting 20 points in several regions of the
painting to quantify the colour difference (E) after the cleaning
operation, following Eq. (2):

E = (L1 − L2 )2 + (a1 − a2 )2 + (b1 − b2 )2 (2)
where the subscript 1 and 2 refers to the value of the CIELab coor-
dinates before and after the cleaning, respectively.
Colour differences were calculated as the Euclidean distance be-
tween the same selected pixel on the CIELab images extracted from
the RIS data cubes before and after the cleaning. L, a, b oscilla-
A. Dal Fovo, S. Mattana, A. Ramat et al. Journal of Cultural Heritage 61 (2023) 91–99

Fig. 2. SC composite images of Panel 1 (a) and Panel 2 (b) combining the single-colour SC maps obtained from the spectral correlation with the respective reference spectra
(c).

Fig. 3. RGB image of Panel 1 (left) with red rectangles corresponding to the magnified areas (right) of the corresponding NIR image at 1292 nm extracted from the RIS
datacube for the visualization of hidden details.

Table 1
Identified pigments composing the main mixtures on the painting.

Targeted region Identified pigment mixture

Architecture walls (S1, S2) Red ochre, raw sienna, orpiment, lead white
Architecture roofs and floor (S1, S2) Natural umber, green earth
Skin tone (S1, S2) Green earth, cinnabar, natural umber, lead white
Clothes of the three figures in the middle (S1) and in the right corner (S2) Natural umber, green earth, bone black
Clothes of the figure on the right (S1) and on the roof (S2) Azurite, lead white, red ochre
Tree foliage (S1, S2) Green earth, natural umber, bone black, lead white
Ground and hillside (S2) Natural umber, raw sienna, green earth
Blanket on the bed (S1) Orpiment, lead white, red ochre, indigo
Bed headboard (S1) Raw sienna, red ochre
S1 = Panel 1, S2 = Panel 2

95
A. Dal Fovo, S. Mattana, A. Ramat et al. Journal of Cultural Heritage 61 (2023) 91–99

Fig. 4. PC composite images of Panel 1 before (a) and after (b) the cleaning, obtained by assigning the first three PCs to the R, G, B components of the tricromatic space;
loadings 1, 2 and 3, plotted as a function of wavelength, before (c) and after (d) the cleaning.

tions around the selected pixel were estimated to be around 3% for Table 2
each coordinate, and attributed to the uncertainties δ L, δ a, δ b. The Colorimetric results on the measured points showing colour differences before and
after cleaning higher than 4.5.
mean square error on E was calculated basing on the standard
error propagation formula following Eq. (3): point time L∗ a∗ b∗ E δE
  2 1 b.c. 48 19 22 7 1.8

n
∂f a.c. 51 23 27
δf = δx (3)
i=1
∂ xi i 3 b.c. 28 −1 9 12 0.8
a.c. 22 9 9
5 b.c. 24 0 1 13 0.8
where f is a generic function of the n variables xi and the symbol
a.c. 16 7 −6
δ indicates the uncertainty for xi . 6 b.c. 64 5 18 7 2.9
Therefore, the error for E, i.e. δ (E ), resulted: a.c. 69 5 13
 2  2  2 10 b.c. 50 0 19 8 1.9
L a b a.c. 45 3 25
δ ( E ) = δL + δa + δb (4) 13 b.c. 69 −2 20 9 2
E E E a.c. 73 −3 12
15 b.c. 46 −3 11 5 1
where L = L1 − L2 , a = a1 − a2 , and b = b1 − b2 . a.c. 44 2 12
The bar chart in Fig. SM3 in the Supplementary Material shows 16 b.c. 51 2 10 6 0.6
the resulting E before and after the cleaning for each measured a.c. 50 3 4
19 b.c. 25 1 0 16 0.7
point.
a.c. 22 14 −9
Points showing colour differences higher than 4.5 are reported
in Table 2. Major differences in colour directions a and b are found b.c. = before cleaning; a.c. = after cleaning.
for points 3, 5, and 19. Specifically, a veering towards red (+a) in
both points 3 and 5, and towards yellow (+b) in point 19.
rable to the instrument axial resolution. No discontinuity between
4.2. Spectral-domain optical coherence tomography (Sd-OCT) the in-painting (right side of the xy section) and the original paint
was detected except for the superficial transparent material that is
OCT analysis was carried out on 12 areas (see the Supplemen- present only on the original paint.
tary material, Fig. SM4) to investigate the stratigraphy and the ar- Results on area 5, including a region where the paint is laid
eas across the original paint and the in-painting. As an example, over the gold leaf and representing the tree foliage, are reported
we report herein the results on area 2 and 5. Area 2 (highlighted in in Fig. SM5 in the Supplementary Material. Five out of the 1430
green in Fig. 5a) encloses a paint integration over a lacuna nearby xz sections (Fig. SM5c) were extracted to better appreciate the de-
the footwear on the right. The xz section (Fig. 5d) is highlighted tails of the stratigraphy. The crack running diagonally across the
in red both in the micrograph (Fig. 5a) and the 3D rendering of examined area, clearly visible in the micrograph (Fig. SM5a) and
the tomocube (Fig. 5b). The intensity depth-profile acquired along in the 3D rendering of the tomocube (Fig. SM5b) can be also vi-
the blue line evidenced by the two-headed arrow in the xz-section sualized in the sequence of xz sections (highlighted by the yellow
is shown in Fig. 5b. The two main intensity peaks are the signals arrows). The two magnifications (Fig. SM5d and e) correspond to
coming from the two outer interfaces encountered by the radia- the areas framed by the red rectangles in section 750/1430 and
tion. The first one is between the air and a semi-transparent layer, 905/1430 and facilitate the visualization of the stratigraphy. In Fig.
which possibly is varnish and whose thickness is around 30 μm SM5d, two interfaces are clearly detected, namely air/paint and
(at the blue vertical line in Fig. 5d). The second one is between the paint/gold, whereas in Fig. SM5e, a signal coming from a surface at
varnish and the original paint Traces of the paint/preparation inter- a higher depth can be observed, likely corresponding to the prepa-
face are slightly visible, possibly due to the small thickness compa- ration layer.

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A. Dal Fovo, S. Mattana, A. Ramat et al. Journal of Cultural Heritage 61 (2023) 91–99

Fig. 5. OCT analysis on area 2: a) micrograph showing the examined area (green square) and the position of the xz section (red line); b) OCT tomocube (5 × 5 × 0.6 mm),
with xz section (red rectangle); c) signal intensity plotted as a function of depth; the two main peaks correspond to the air/paint and paint/preparation interfaces; d) xz
section allowing for the visualization of the stratigraphy.

Fig. 6. Results of time-resolved fluorescence imaging, phasor, and GMM analysis of area 3: a) RGB image of the detail of Panel 1, the analysed area highlighted by the red
rectangle; b) τ -phase map superimposed to the white light image of the areas of interest; c) phasor plot showing the clusters; d) clustering of the phasor clouds after the
application of the GMM model; e) segmented cluster GMM map superimposed on the RGB image.

3.3. Fluorescence lifetime imaging (FLI)
FL mapping was performed on several areas over the surface
of Panel 1, specifically in the regions across the paint integration
and the original paint where OCT analysis was also carried out.
Here we show the results of areas 9 and 3 (refer to Fig. SM4 in
the Supplementary Material). For each measurement, each τ -phase
was computed as an average over 30 0 0 acquisition points with an
integration time of 15 ms each. Results on area 9 are reported in
Fig. SM6 in the Supplementary Material. Measurements were per-
formed in the region highlighted in red in Fig. SM6a. In this case,
phasors corresponding to the original paint and the paint integra-
tion were found to aggregate in two partially overlapping clusters
within the phasor plot (Fig. SM6d), in which the density colour
scale corresponds to the number of decays having the same pha-
sors. The measured τ -phase of the in-painting retouching (clus-
ter 1) and the original paint (cluster 2) are 2.11 ± 0.13 ns and
1.68 ± 0.15 ns, respectively. Each point of the phasor cluster can
be directly related to the τ -phase map (Fig. SM6b) due to the
reciprocity between the fluorescence decay and the correspond-
ing phasor, upon Fourier transformation. To identify the cluster to
which each phasor point pertained in an automatic and unsuper-
vised manner and to assign a probability to each phasor per clus-
ter, we applied the GMM method using K = 2. At each spatial coor-
dinate, the corresponding phasor was assigned to the cluster with
the higher probability resulting from the GMM analysis. Segmen-
tation of the two cluster was then realized, based on the maxi-
mum probability assigned to each phasor, as shown in the GMM
plot (Fig. SM6e), where red and green colours are used to identify
the cluster 1 and 2, respectively. Finally, the spatial location of each

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A. Dal Fovo, S. Mattana, A. Ramat et al.
Table 3
FL phasor analysis of area 3.
Cluster τ -phase (ns) St. Dev. (ns) St. Error
1 - Blue 2.29 0.12 0.004
2 - Red 2.50 0.22 0.009
3 - Green 1.69 0.21 0.006
cluster was retraced to the pixels of the RGB image (Fig. SM6c) al-
lowing to clearly visualizing the distribution of the two materials
based on their phasor.
Area 3 (Fig. 6a) includes three different materials, namely the
green floor (original paint and in-painting integration), and the
brown clothing of the figure. While the phasor map (Fig. 6b) re-
sulting from the phasor analysis (phasor cluster reported in Fig. 6c)
does not allow for a clear identification of the three fluorescent
paints, the application of the GMM model with K = 3 provides a
good discrimination of the cluster distribution (Fig. 6d). The green
original paint (cluster 1), the brown clothing (cluster 2) and the
green retouching (cluster 3), are represented in blue, green and
red, respectively, and are reported with the corresponding mean
τ -phase in Table 3. The presence and location of the three com-
ponents over the painting surface are identified by the GMM map
superimposed to the RGB image in Fig. 6e.
4. Discussion and conclusions
The results from the combined application of complementary
imaging techniques confirmed the advantages of using a multi-
analytical approach for an extensive study of paintings, which was
the objective of this study.
Spectral analysis of the reflectance imaging dataset, integrated
with information previously obtained with XRF, allowed defining
the pigment palette used by Lorenzetti for two panels of the al-
tarpiece. The set of egg tempera samples, specifically prepared ac-
cording to historical recipes, proved suitable as a reference for a
direct comparison with the painting under investigation. All pig-
ments were identified in both panels, except for indigo, which was
used only in Panel 1. The distribution of each pigment was mapped
with a good level of confidence based on spectral correlation, en-
abling to identify the location of the main pigment mixtures over
the paintings’ surface. Regions not covered by the SCM mapping
(black areas in the combined SC images, such as part of the ground
in Panel 2) are due to the selection of the 0.95–1 spectral similarity
range, which only includes pixels with high similarity to the refer-
ence pigment. These values are attributable to pigments present in
high relative quantities within the mixture. On the contrary, the
spectral characteristics of pigments present in traces or covered by
other paint layers resulted in spectral similarity values lower than
the selected window, and, thus, were excluded from the mapping.
The analysis of RIS images in the NIR range allowed visualizing
details not perceivable with the naked eye in Panel 1, such as the
area of the tree foliage painted over the golden leaf, a pentimento
in the architecture, and the paint integration on the figure on the
right.
OCT measurements allowed identifying the composing layers
and quantifying micrometric features in the painting stratigraphy.
Finally, the phasor analysis of FL data proved suitable to rep-
resent the fluorescence lifetime measured from molecular micro-
environmental heterogeneity, such as the original paint and in-
painted areas, as clusters of fluorescence lifetime distributions in
the phasor domain. The measured phasors are representative of
the whole paint system (i.e. pigment + binder) as a result of
the interactions between the fluorophores and the non-fluorescent
species. We assume that the overall fluorescence signal from the
paint layer is strongly influenced by the binder in which the pig-
ment particles are dispersed, especially if the pigment is a weak
98
Journal of Cultural Heritage 61 (2023) 91–99
emitter or does not exhibit any fluorescence. Areas characterized
by sufficiently different fluorescence lifetimes resulted in spatially
separated clusters within the phasor space, thus enabling the visu-
alization of multiple compounds. The measured differences in the
fluorescence decay are related to the molecular environment and
the relative amounts of fluorescent species, which can greatly dif-
fer from the original paint and the paint integration, thus substan-
tially affecting the measured τ -phase. The use of GMM algorithm
offered the advantage to automatically segment the images in the
phasor domain rather than manually select each region in the real
space domain, by assigning a probability to each phasor cluster.
This automatic approach improves the reproducibility of the exper-
iment and enables to extract quantitative and impartial data from
the experimental dataset.
Funding
This research was supported by IPERION HS – Integrating Plat-
forms for the European Research Infrastructure ON Heritage Sci-
ence Project (DFM.AD006.166).
Acknowledgements
Dr. Cecilia Frosinini and Dr. Sandra Rossi of the Opificio delle
Pietre Dure are gratefully acknowledged for their interest in this
research and the fruitful discussion on the results of the analysis.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.culher.2023.03.002.
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