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Cross­Section and Staining­Based Techniques for Investigating Organic

Materials in Painted and Polychrome Works of Art: A Review
Irina Crina Anca Sandu, Stephan Schäfer, Donata Magrini, Susanna Bracci and Cecilia A. Roque

Microscopy and Microanalysis / Volume 18 / Issue 04 / August 2012, pp 860 ­ 875

DOI: 10.1017/S1431927612000554, Published online: 31 July 2012

Link to this article: http://journals.cambridge.org/abstract_S1431927612000554

How to cite this article:

Irina Crina Anca Sandu, Stephan Schäfer, Donata Magrini, Susanna Bracci and Cecilia A. Roque (2012). Cross­Section and
Staining­Based Techniques for Investigating Organic Materials in Painted and Polychrome Works of Art: A Review.
Microscopy and Microanalysis,18, pp 860­875 doi:10.1017/S1431927612000554

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Microsc. Microanal. 18, 860–875, 2012
doi:10.1017/S1431927612000554 Microscopy AND

Microanalysis
© MICROSCOPY SOCIETY OF AMERICA 2012

REVIEW ARTICLE
Cross-Section and Staining-Based Techniques for
Investigating Organic Materials in Painted and
Polychrome Works of Art: A Review
Irina Crina Anca Sandu,1, * Stephan Schäfer,2 Donata Magrini,3 Susanna Bracci,3 and
Cecilia A. Roque 4
1
REQUIMTE, Departamento de Conservação e Restauro, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa,
Campus de Caparica, 2829-516 Caparica, Portugal
2
Departamento de Conservação e Restauro, Faculdade de Ciências e Tecnologia (FCT), Universidade Nova de Lisboa (UNL),
2829-516, Caparica, Portugal
3
Institute for Conservation and Valorization of Cultural Heritage (ICVBC), National Council of Research (CNR),
via Madonna del Piano 10, 50019 Sesto Fiorentino, Florence, Italy
4
REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia (FCT), Universidade Nova de Lisboa (UNL),
2829-516, Caparica, Portugal

Abstract: The article presents a review of the use of cross-section and staining techniques for investigating
natural organic materials ~mainly proteinaceous and oil-based binders/varnishes! in painted and polychrome
artworks, considering the requirements of conservation practice and routine diagnostics. The reviewed
literature calls attention to the importance of using cross sections to prepare samples for optical microscopy and
to different properties of embedding resins; the most appropriate instrumental conditions for optical micros-
copy; and the advantages and disadvantages of the most common staining techniques. A few case studies were
selected to illustrate the use of autofluorescence ~intrinsic fluorescence! and induced fluorescence ~using specific
staining tests and fluorophore-labeled antibodies! for mapping and identifying organic paint materials in cross
sections. New directions of research in cross-section analyses and fluorescence-based techniques for the
identification and mapping of artistic materials are presented. The complementary use of different stains on the
same cross section, further exploration of intrinsic and induced fluorescence of aged versus fresh materials, and
applicability of cross-section observation and staining as complementary methods for assessing the effectiveness
of restoration treatments, such as cleaning and consolidation, are discussed in the last section of the article.
Key words: cross section, staining tests, natural organic materials, conservation practice, routine diagnosis,
painted/polychrome works of art

I NTR ODUCTION
Several analytical techniques, used in investigating painted
and polychrome works of art, can provide answers to ques-
tions raised by art conservators, curators, or art historians.
Determining the authenticity of a work of art, identifying
its material composition, the artistic technique and past
restorations, and the effects of the interactions with various
exogenous factors are the main goals of a routine diagnostic
project or investigation ~Aldrovandi & Picollo, 2001; Appo-
lonia & Volpin, 2001; Sandu et al., 2009a!.
The oldest basic and most readily available method that
give insight into the stratigraphic structure and composi-
tion of a polychrome or paint sample is the study of cross
sections and eventually of thin sections obtained of it ~Plest-
ers, 1956; Jones, 1962; Matteini & Moles, 1984; Tsang &
Cunningham, 1991; Messinger, 1992; Derrick et al., 1994;
Martin, 1994!. Cross-section-based methods for observing
and analyzing organic materials present several advantages
~Tsang & Cunningham, 1991; Khandekar, 2003; Sandu et al.,
2008!; for daily use in scientific and conservation laborato-
Received September 23, 2011; accepted April 11, 2012
*Corresponding author. E-mail: irina.sandu@fct.unl.pt
ries such as spatial resolution versus bulk methods, the
possibility of mapping the organic materials in each layer of
the stratigraphic structure and distinguishing different ma-
terials according to their intrinsic fluorescence. These meth-
ods are simple and require readily available equipment
~Spring & Davidson, 2008!. However, they have technical
limitations and drawbacks such as the micro-invasiveness
and the need to have a perfectly flat surface of the cross
section in order to achieve proper focus and obtain clear
images.
Fluorescent stains have been an alternative to the much
older stains ~such as Amido Black, Acid Fuchsine, Ponceau
S, Stain-all, Biuret, etc.! for analyzing and identifying bind-
ing media on cross sections for over 20 years ~Plesters, 1956;
Gay, 1970, 1978; Martin, 1977, 1978; Matteini et al., 1981;
Spring, 1991!. They were initially popularized by R. Wolbers
~Wolbers & Landrey, 1987; Wolbers, 1990, 2000!, who intro-
duced them as an aid in selecting cleaning methods and for
characterizing binding media in painting cross sections. In
addition to fluorescein isothiocyanate ~FITC!, rhodamine,
and other fluorescent stains ~Nairn, 1962; Maeda et al.,
1969; Green et al., 1973; James & Tas, 1984; Banks &
Paquette, 1995! used as alternatives to traditional dyes,

Figure 1. Schematic structure and composition of a painted or polychrome sample.
other products with different fluorophore groups ~DyLight,
Nile Red, SYPRO! have been used in biological labeling and
protein staining ~Holmes & Lantz, 2001; Patton, 2002; Wang
et al., 2006; Sandu et al., 2012!.
The main purpose of the present article is to review
the readily available optical microscopy methods for investi-
gating organic materials in painted and/or polychrome works
of art ~Mills & White, 1979!. A few case studies draw atten-
tion to the use of cross sections in conjunction with staining-
based techniques as a simple, reliable method for mapping
and identifying artistic organic materials ~mainly protein
and oil-based materials!. Additionally, the different proper-
ties of embedding resins for obtaining cross sections, the
main staining techniques for organic materials, and alterna-
tive fluorophore-labeled antibody techniques such as immu-
nofluorescence microscopy ~IFM! for protein detection are
discussed and illustrated. Some of these methods were origi-
nally developed for other more complex analytical tech-
niques @e.g., confocal laser scanning microscopy ~CLSM!,
proteomics, etc.#, but they have been successfully tested for
use with optical microscopy in cultural heritage applications.
Several other imaging techniques applied on cross
sections @such as micro-FTIR, attenuated total reflectance
~ATR!-FTIR, micro-Raman, chemiluminescence, etc.# are
not covered, and just proteinaceous and lipidic binders are
considered in this review.
New directions of research to identify and to map
organic material are also suggested, as the complementary
use of different stains on the same cross section.
The need to better explore the fluorescence of aged
versus fresh materials and the usefulness of microscopic
observations and staining to evaluate the efficiency of resto-
ration treatments are briefly discussed.
N ATURAL O RGANIC M ATERIALS IN
P OLYCHR OME S AMPLES AND
T HEIR I DENTIFICATION
Natural organic materials ~Matteini & Moles, 2002;
Doménech-Carbó, 2008! used in polychrome works of art
are commonly grouped in the following categories: protein-
Cross-Section and Staining Techniques for Art 861
aceous ~egg, fish, and other animal glues, casein!, polysac-
charide ~gum arabic, tragacanth, fruit-tree gums, starch,
and dextrin!, lipid-based ~drying oils such as linseed, poppy
seed, walnut oils, etc.!, resins, and waxes ~Slansky, 1956;
Gettens & Stout, 1966; Kühn, 1986; Rinuy & Gros, 1989;
Masschelein Kleiner, 1992; Mills & White, 1994; Sandu
et al., 2005; Petty, 2007!. These materials can be found alone
and as mixtures with mineral phases ~inert materials! or
pigments to form polychrome or protective layers ~Fig. 1!.
Sometimes, binary or even tertiary mixtures, such as tem-
pera grassa in which oil is added to egg yolk, have been
found. In some cases, binders or consolidant materials, such
as egg white, isinglass, or animal glues, were also used as
coatings or temporary varnishes ~Kühn, 1986; Phenix, 1997;
Carlyle, 2001; Matteini & Moles, 2002!.
If the identification of inorganic constituents of poly-
chrome layers ~pigments, inert materials, fillers! is consid-
ered affordable, using standard conservation science tools,
the characterization of organic materials still represents a
challenge for conservation scientists. Difficulties relating to
identification and mapping binders in the everyday conser-
vation practice can be explained by the following reasons
~Messinger, 1992; Rosi et al., 2011!:
• the low content ~micrograms or even nanograms! of
organic materials generally encountered in heterogeneous
micrometric samples
• the presence of very thin layers and mixtures of different
materials, especially in the case of overpainted or restored
objects, when original materials are present together with
new materials in the same sample and/or layer
• the interference from inorganic materials or phases that
can cause problems in properly visualizing the organic
materials in cross sections
• aging and degradation/denaturation processes that usu-
ally lead to a loss in properties such as fluorescence and
solubility of the organic materials that could be useful in
their identification ~Karpowicz, 1981!
• the lack of knowledge of the conservation history of an
object or sample can lead to erroneous interpretations of
information that the cross-section and staining tests could
offer.
862 Irina C.A. Sandu et al.
C R OSS S ECTIONS AND T HEIR S TUDY
It is quite usual for conservators and restorers to consider a
painting as a stratified structure. For this reason, several
analytical methods have been developed for studying the
internal layers of a painting, and many of them are using a
cross section as a support for analysis. Before any analytical
technique is applied on the cross section of a sample, we
must be sure that sampling was performed carefully, respect-
ing the integrity of the work of art and that the sampling
areas are representative for the final purpose of the scientific
study. Therefore, the sampling step is crucial for excluding
different sources of error or contamination to narrow the
range of choices for reliable identification and mapping of
these materials. A common problem arises, for example,
when samples are taken in previously restored areas of an
art object where the original paint composition has changed.
However, by applying noninvasive imaging techniques
@photographic techniques using ultraviolet ~UV! fluores-
cence and infrared ~IR! reflectography, fiber optics fluores-
cence spectroscopy, etc.#, the conservator-restorer is able to
choose an area that is highly representative of the object’s
entire structure and its history for a correct collection of
data ~Matteini & Moles, 1984; Sandu et al., 2005, 2009b!.
To perform proper microscopic examination, the paint
fragment must be placed on edge, must be mounted on a
suitable medium, and smoothly polished to give a plane
surface for focusing under the microscope. The paint sec-
tion provides a large amount of precise information from a
very small area of the painting. The preparation of paint
cross sections was begun more than 40 years ago ~Plesters,
1956!, and the conservation literature contains many stud-
ies focused on observations of cross sections of paint/
polychrome samples with an optical microscope under visible
and UV light ~Ploem & Tanke, 1987; Becker, 1989; Rinuy &
Gros, 1989; Siegel, 1993; Martin, 1994; Dorge & Carey
Howlett, 1994; Derndarsky & Ocklind, 2001; Sandu et al.,
2005! and as support for more elaborate analyses. Among
these, we must mention scanning electron microscopy ~Pinna
et al., 2009; Sandu et al., 2010!, X-ray fluorescence ~Pinna
et al., 2009!, micro and fluorescence spectroscopy ~White,
1984; Osmond, 1993; Pinna et al., 2009; Rosi et al., 2011!,
microRaman, microFTIR, and ATR-FTIR ~Doménech-Carbó,
2008; Pinna et al., 2009!, immunofluorescence assays ~Tal-
bot, 1982; Kockaert et al., 1989; Ramirez-Barat & de la Vina,
2001; Heginbotham et al., 2004; Vagnini et al., 2008; Pinna
et al., 2009; Sandu et al., 2009b; Cartechini et al., 2010;
Arslanoglu et al., 2011; Sciutto et al., 2011!, and staining
tests ~Feigl & Anger, 1966; Gay, 1970, 1978; Martin, 1977,
1978; Rinuy & Gros, 1989; Wolbers, 2000; Sandu et al., 2006,
2012!.
Many stains ~dyes! have been used since the early stages
of scientific conservation and investigation ~Plesters, 1956;
Gay, 1978; Martin, 1978; Wolbers & Landrey, 1987; Wolbers,
1990! for mapping and identifying organic components
~Matteini et al., 1981; Matteini & Moles, 1984; Mills &
White, 1994; Schäfer, 1997; Petraco & Kubic, 2004; Sandu
et al., 2006! such as binders, sizes, varnishes, adhesives, and
consolidating materials ~Kühn, 1986; Matteini & Moles,
2002!. However, even though they are quite versatile and do
not require a highly technological setup, they have fallen
out of favor due to a lack of specificity, interference prob-
lems, or because other methods have become available.
Recently, however, there has been a revival of interest in
using the cross-section method as a support for identifying
and mapping binders; there is a great deal of research to be
done concerning the simultaneous identification of differ-
ent classes of organic materials such as oil/fatty and protein-
based binders in the same cross section.
Preparing Cross Sections for Optical Microscopy,
Staining Tests, and Immunological Methods
Cross-section analysis is a method of embedding a paint or
polychrome sample in a synthetic resin to form a rigid and
transparent block for visualizing the stratigraphic sequence
of layers within the sample. Derrick et al. ~1994! describe a
wide range of embedding media used in conservation
and also in forensic or biomedical applications ~polyester,
epoxy, acrylics, cyanoacrylates, gelatin, wax, hot-melt
adhesives, and silicones!. Pilc and White ~1995! describe the
use of silver chloride for embedding samples prior to thin
sectioning for IR microscopy. The main types of commer-
cially available resins can be grouped in cold systems ~such
as room temperature curing epoxy! and hot systems ~such
as phenolic resin powder cured in a high-pressure device
at 140–2008C! ~Wachowiak, 2004!. Since samples from the
cultural heritage context are often heat sensitive ~mostly
those made of organic materials!, it is best to eliminate
hot systems. Chemically, the embedding resins can be
grouped as the thermoset polymers ~epoxy, polyester! and
thermoplastic polymers ~Canada balsam, catalyzed acrylic,
cyanoacrylate!. The thermoset ones generally have better
performances because they are more abrasion, solvent, and
temperature resistant. Between the cold system thermoset
resins, epoxies may be the best choice, although Waentig
~1993! concluded that both polyester and epoxy would be
useful. Derrick et al. ~1994! discussed methods to improve
polyester embedding for microtomy and binder analysis.
Bousfield ~1992! supplies very detailed descriptions of both
preparation method and materials selection, to the point
that he defines auditable laboratory procedures. Because of
general performance qualities, Bousfield indicates that ep-
oxy is preferable over polyester resin.
Generally, the embedding resins used for obtaining a
cross section from a paint sample should have the following
characteristics: readily available on the market; uniformity
from one batch to another; low viscosity as a monomer for
penetration; uniform polymerization; little volume change
during polymerization; good preservation of the fine struc-
ture; good sectioning quality that includes homogeneity,
hardness, plasticity, and elasticity; resistance to the heat
generated by sectioning; adequate specimen stainability;
and stability under the electron beam and at high vacuum
conditions. Epoxy resins have optical properties suitable for
 Cross-Section and Staining Techniques for Art 863

Figure 2. Images of cross sections with different embedding resins @~a! polyester resin, ~b! acrylic resin, and ~c! epoxy
resin# acquired with an optical microscope ~images in reflected visible light in the first row; fluorescence images with UV
excitation using filter set 365/420 nm in the second row; and visible excitation using filter set 450/515 nm in the third
row!.

immunofluorescence ~Vagnini et al., 2008! as they do not
swell, are stable to aqueous solvents, and have a good
transparency in the IR spectral range for integrated IR
spectroscopic techniques ~Heginbotham et al., 2004!. Epoxy
resins perform best in common electron microscopy, while
polyester resins are considered the best option for optical
microscopy and immunofluorescence, however with the
drawbacks of infiltrating porous samples and low binder
content samples ~Derrick et al., 1994!.
Cross-section samples may be cut with a microtome to
obtain numerous thin sections ~Martin, 1978; Tsang & Cun-
ningham, 1991!, or they may be polished on opposite sides
to yield a single thin section ~Garrido & Cabrera, 1986!. A
thickness of 5–50 mm is commonly reported for thin sec-
tions in the conservation literature.
Figure 2 shows images in visible reflected light ~first
line! and fluorescence images of paint samples embedded in
three different resins ~polyester, acrylic, and epoxy!.
Traditional Staining Tests
Around 1905 Oswald for the first time performed analyses
on cross sections using biological stains ~Spring, 1991!.
Since then, a variety of stains ~with visible or fluorescent
emission range! have been suggested in the conservation
literature ~Gay, 1970, 1978; Johnson & Packard, 1971; Mar-
tin, 1978; Matteini et al., 1981; Wolbers & Landrey, 1987;
Rinuy & Gros, 1989; Wolbers, 1990, 2000; Sandu et al.,
2009b, 2012!. The staining technique is mainly based on the
use of dyes able to form colored compounds with organic
materials, such as proteins, polysaccharides, resins, and oils.
864 Irina C.A. Sandu et al.
When applied to paint cross sections, the accuracy of the
test depends on the selectivity of the dye used and is greatly
influenced by the sample’s age and by the dye absorption
phenomena that may take place on the surface. The detec-
tion limit of the methods varies significantly, but generally
ranges within a few micrograms of the unknown compound.
Limitations in the formation of the staining color can
be due to dye absorption by porous matrices ~such as
calcium carbonate grounds! or to the degree of aging/
denaturation of the materials to be identified. Although
aging could give a decrease in the reactivity of the organic
materials, in some cases denaturation processes, leading to
an increase in the number of functional groups available for
the dyeing, favor the formation of the staining color ~Mar-
tin, 1977; Matteini et al., 1981!. The application of staining
tests to samples from paintings and other polychrome works
of art usually requires that the operator be familiar with the
artist’s technique and materials. Staining tests always require
controls with both fresh and aged materials ~already chem-
ically characterized!, before assuming that they will work
unambiguously on unknown samples.
The identification of proteinaceous compounds through
histochemical ~staining! tests is based on the interaction
with specific functional groups ~such as carbonyl, carboxyl,
hydroxyl, etc.! and/or on the characterization of specific
properties of chemical functions of these materials: redox,
acid-basic, or metachromatic properties ~Feigl & Anger,
1966; Gay, 1970; Matteini et al., 1981; James & Tas, 1984;
Matteini & Moles, 1984!.
Many protein stains, such as Acid Fuchsine and Amido
Black, are acidic dyes. The interaction of the dye with
proteinaceous paint materials will depend on the pH of the
stain solution because this determines the protein’s net
charge. The presence of dark or colored pigments can
complicate the identification of the positive stain especially
if it is the same color as the pigmented layer. Some of the
dyes, such as the acidic ones, may also dissolve salts or inert
charges, as in the case of calcium carbonate.
The histochemical dyeing of lipids ~triglycerides, phos-
pholipids, cerides! in paint or polychrome samples is based
on reactions with formation of chromophore groups using
lysochromic dyes capable of dissolving the lipids and/or
specific reactions for some radicals ~such as carbonyl from
the ketone and aldehyde groups! or reactions to prove the
unsaturated state of an acid ~Matteini et al., 1981!. The
staining tests for lipids are more difficult to evaluate as they
are less homogenous, less intense, and/or less stable than
the protein stains.
Table 1 lists the most reported traditional stains and
their preparation together with the specific positive re-
sponses for organic paint materials.
Acid Fuchsine ~Fuchsine S! was first suggested by
Plesters ~1956! as an acidic dye that, by means of electro-
static interactions, gives a positive staining red-pink or dark
red color for gelatins and animal glues, and pink for egg
white and egg yolk proteins. The strength of the staining
color depends on the number of amino groups available for
bonding and will therefore vary between different proteins.
Gay ~1970, 1978! found that it also stains starch, even
though other stains ~such as Lugol! have proven better in
identification of starch and polysaccharides. Acid Fuchsine
can give a fluorescence emission when bonded to egg
protein, although less intense when bonded to glue ~Gay,
1978!.
Amido Black, an azoic dye ~Blue/Back Naphtol 10B!
introduced by Martin ~1975, 1977, 1978! is one of the
common stains currently used in conservation for distin-
guishing different proteinaceous materials. The blue posi-
tive staining is due to an acid-basic reaction with a proteins’
functional groups. It can be formulated in three different
solutions according to the pH ~AB1, acidic; AB2, moderate
acidity; AB3, neutral! that can be used to obtain some
differentiation between various proteins ~Martin, 1975, 1977!.
Martin ~1978! claimed that its specificity was such that it
was possible to identify emulsions on a cross section, and
that there were staining differences between protein-oil and
oil-protein emulsions. This stain makes it possible to distin-
guish between egg and glue, and the staining of egg proteins
is stronger than the rather weak, ambiguous color obtained
with Acid Fuchsine.
Ponceau S stain forms bonds with proteins similar to
those obtained with Amido Black and Fuchsine. Johnson
and Packard ~1971! first introduced this stain, which gives a
bright pink-red to glues and a bright opaque orange red to
egg yolk-containing samples. This stain can identify pro-
teins more precisely, although with subtle differences in
hue. The authors consider the opaque staining as an advan-
tage, but it may in fact be indicative of nonspecific staining.
They also claimed that they could identify emulsions by first
staining with Ponceau S that gives an uneven color and then
with an oil stain such as Sudan Black B. Ponceau S stains old
paint samples more strongly than fresh ones, as might be
expected, because denaturation results in more amino groups
being available for bonding. Its drawback is that it may react
also with Ca 2⫹ present in painting layers ~Moles et al., 1982;
Matteini & Moles, 1984!.
“Stains-all” is a cationic carbocyanine dye that has
been used in biochemistry, its first application on paint
samples being suggested by White ~1984!. It stains proteins
with different colors according to their phosphorous con-
tent, which is characteristic for each proteinaceous paint
binder. As a cationic dye, it interacts with carboxylate groups
on the protein rather than amino groups. Once the bond is
formed, the aggregation of the dye molecules and therefore
the color depends on the amino acid sequences in the
protein. In biochemical applications it was found to stain
nonphosphorylated proteins red and phosphorylated pro-
teins, such as casein, blue ~Green et al., 1973!. It also has
some disadvantages, such as extremely quick fading under
the light so that samples have to be kept in the dark. It also
stains other macromolecules such as nucleic acids and poly-
saccharides; therefore, it is not as specific as other stains
already in use. Denaturation of the protein may affect
interactions. Also, different factors, such as pH and the
 Cross-Section and Staining Techniques for Art 865

Table 1. Common Stains for Identifying Organic Paint Materials.

Positive Response and the
Staining Agent Preparation Specific Color of the Stain
Acid Fuchsine The test is done by placing a drop of agent ~aqueous solution Gelatins and animal glues are col-
~Fuchsine S! 1%! for 10–15 min on the sample, then washing with a solution ored dark red, while egg white
of acetic acid/water ~1/99 v/v! ~Gay, 1978; Spring 1991!. and egg yolk are colored light
pink.
Amido Black 1 g of agent is dissolved in 450 mL of acetic acid, diluted in 450 A dark blue color indicates the
AB1 mL of sodium acetate 0.1 M and 100 mL glycerin. The solution presence of egg yolk.
is left to react for 5 min on the cross section and then washed
with a 5% aqueous solution of acetic acid ~Martin, 1977; Spring,
1991!.
Amido Black 1 g of agent is dissolved in 450 mL of acetic acid diluted in 450 A dark blue color indicates the
AB2 mL solution of sodium acetate 0.1 M and 100 mL glycerin. The presence of aged proteins of ani-
solution is left to react for 5 min on the cross section and then mal glues and casein.
washed with a 1% aqueous solution of acetic acid ~Martin,
1977; Spring, 1991!.
Amido Black 1 g of agent is dissolved in 900 mL water and 100 mL glycerin. A blue color indicates the pres-
AB3 The solution is let to react for 5 min on the cross section and ence proteins of gelatins and
than washed with a 1% aqueous solution of acetic acid ~Martin, casein.
1977; Spring, 1991!.
Ponceau S The agent must be prepared the day before to obtain a saturated An orange-red color identifies egg
solution of colorant in acetic acid 3%. For the test, a drop of yolk, while a red-pink color indi-
solution is applied to the cross section, left to react for 20 min cates the presence of the animal
and then washed with water ~Johnson & Packard, 1971!. glue.
Note: Consider also that Ca 2⫹ containing materials ~carbonate
or gypsum! give colorations with Ponceau S at different pH,
with the formation of an insoluble red complex.
Stains-all ~1! 0.005 g in 100 mL isopropyl alcohol/water mixture 25/75 v/v Nonphosphorus proteins turn red;
~purple color; pH 3!—good for differentiating between proteins. casein and proteins with phos-
~2! Saturated solution in isopropyl alcohol/water mixture 25/75 phorus content greater than 0.2%
v/v ~reddish color!—this formulation gives a good differentia- turn blue.
tion between proteins but of entirely different colors ~e.g., Polysaccharides turn blue/purple.
dark red for egg and green for gelatin! ~White, 1984; Spring,
1991!.
Oil Red O 0.125 g of agent is mixed with 25 mL isopropyl alcohol and after An intense red color indicates the
1 h the solution is filtered. Dilute 6 mL of stock solution with 4 presence of oils and waxes.
mL distilled water. Place a drop on the cross section and let react
for 10 min. Then wash with isopropyl alcohol/water 60/40 v/v
for 3–5 s and then with distilled water ~Matteini et al., 1981!.
Sudan Black B The dried films of lipidic binders cannot dissolve the lysoch- A blue or black color indicates
rome colorant, and therefore the test should be done by heating the presence of lipids.
the sample in a closed quartz tube at 708C. The reaction will last
from 5 to 30 min at 708C, with an ethyl alcoholic saturated
solution of Sudan Black B, then washing with ethyl alcohol at
608C is done.
If the test is done with a solution of propylene glycol, the
reaction will last 5–7 min, using a solution of 0.7 g Sudan Black
B in 100 mL propylene-glycol, followed by a washing with a
solution of propylene-glycol/water ~85/15 v/v! ~Matteini & Moles,
1984!.
Lugol’s solution Solution available in different iodine concentrations: 1%, 2%, or A dark-blue/black color indicates
5%. the presence of starches.
The 5% solution consists of 5% ~wt/v! iodine ~I2 ! and 10%
~wt/v! potassium iodide ~KI! mixed in distilled water and has a
total iodine content of 130 mg/mL ~Matteini & Moles, 1984!.
866 Irina C.A. Sandu et al.
Figure 3. Example of staining using Oil Red O on a cross section from an oil paint replica.
solvent in which it is dissolved, greatly affect the behavior of
this dye ~Kay et al., 1964!.
Oil Red O is a lipophilic dye ~Matteini et al., 1981;
Koopman et al., 2001; Beaudoin, 2004! that stains lipids
~oils! and waxes in the paint layers with an intense red
~Fig. 3!. The stain is quick and easy to apply. A stock
solution in isopropyl alcohol has to be prepared and diluted
with water before staining.
Sudan Black B is a lysochrome ~fat-soluble dye! diazo
dye used for hystochemical staining of neutral triglycerides
and lipids on frozen sections and of some lipoproteins on
paraffin sections. The dye is delivered originally as a dark
brown-black powder and yields a blue-black stain solution
with maximum absorption at 596–605 nm ~Matteini &
Moles, 1984; Sandu et al., 2006!.
Lugol’s iodine ~Lugol’s solution! was first made in 1829
as a solution of elemental iodine and potassium iodide in
water, being often used as an antiseptic and disinfectant for
emergency disinfection of drinking water and as a reagent
for detecting starches ~and dextrin! in routine laboratory tests.
Elemental Lugol’s solutions stain starches through iodine’s
interaction with the coil structure of the polysaccharide ~Mat-
teini & Moles, 1984, 2002; Sandu et al., 2006; Barton, 2007!.
Fluorescent Staining Techniques
Fluorescence is caused by the absorption of radiation by the
molecules in the organic materials ~proteins, oils, or resins!
followed by emission at longer wavelength ~de la Rie, 1982;
Ploem & Tanke, 1987; Becker, 1989; Messinger, 1992;
Siegel, 1993; Petraco & Kubic, 2004; Pinna et al., 2009!.
Microscopical observations of the intrinsic fluorescence,
originated by the natural fluorophores in the paint binding
materials, have been widely reported in the routine exami-
nation of works of art ~de la Rie, 1982; Matteini & Moles,
1984; Spring & Davidson, 2008; Pinna et al., 2009!. For
example, protein fluorescence is generally excited by using
UV radiation with wavelength ranging from 280 to 365 nm
~Nairn, 1962; Sandu et al., 2012!, and the emissions are
mostly due to the excitation of tryptophan residues, with a
lower contribution from tyrosine and phenylalanine. Addi-
tional fluorescence is developed after amino-acid degrada-
tion by age-related cross linkage and Maillard reaction
products ~Deyl et al., 1999!.
Reactive dyes or fluorophores can be used to mark or
indicate specific materials with fluorescent dyes as a second-
ary or additional phenomenon ~Wolbers, 1990, 2000!. Sev-
eral reactive secondary fluorochromes were introduced into
the field of art conservation several years ago, starting from
biological science protocols and new ones are being experi-
mented with today, mainly for the detection and mapping of
proteinaceous paint materials in cross-section specimens
~Wolbers, 2000; Sandu et al., 2012!. Proteomic applications
to fluorescence detection of protein-based materials are par-
ticularly useful and have opened new research paths for de-
veloping staining protocols. The linear dynamic range of
detection using fluorescent stains is usually superior to color-
imetric or reverse stains ~Nairn, 1962; Sandu et al., 2012!.
Most of the fluorochromes are not used in aqueous
solution but are dissolved in organic solvents and interact
with proteins by forming covalent bonds. Functional groups
available for tagging fluorochromes with proteins include
amino or carboxyl groups, thiols as well as imines, together
with the amino acid cysteine. The three main kinds of
chemically reactive groups are acid chlorides, isocyanates,
and diazonium salts ~Schäfer, 1997; Holmes & Lantz, 2001!.
Fluorescent stains offer some advantages over visible stains
detectable under visible radiation: since they are about 100
times more sensitive they can detect materials at lower con-
centrations ~Nairn, 1962!; they generally create strong cova-
lent bonds ~Wolbers, 2000!; they are often delivered in

Table 2. The Most Common Fluorochrome Dyes, Used for Staining Binding Media in Paints and Polychrome Works
of Art ~Wolbers, 2000; www.invitrogen.com/Molecular Probes!.
lexc
Fluorochrome ~nm!
Fluorescamine ~FLUR! 390
Fluorescein isothiocyanate ~FITC! 495
~425–525!
Rhodamine B 540
~RHOB! ~430–550!
Tetramethyl rhodamine-isothiocyanate 540
~TRITC!
Lissamine rhodamine B sulfonyl 570
chloride ~LISSA!
Nile Red 485
~552/636 in methanol!
DyLight 488 493
BODIPY 493/503 490–500
SYPRO Ruby 250–350
350–525
SYPRO Orange 250–360
380–600
solvents, avoiding dissolution of the proteins with aqueous
stains ~Banks & Paquette, 1995!; the presence of different
pigments does not influence the results ~Rinuy & Gros, 1989;
Schäfer, 1997; Sandu et al., 2012!. However, fluorescent stains
also have drawbacks such as fluorescence quenching due to
some pigments or the autofluorescence of binding media
overlapping the autofluorescence of the stain and finally the
solubilization of natural oils or resins, which may occur due
to the organic solvent in which the stain is applied.
Table 2 gives the main fluorochromes used as stains for
binding media with their excitation and emission wave-
lengths ~l! and colors and also with the type of materials
they are supposed to react/identify.
F LUOR ESCENT S TAINING TESTS
FOR P R OTEINS
There are several fluorescent dyes that were brought over to
art conservation from proteomics to detect protein-based
materials in paint cross sections ~Udenfriend et al., 1972; De
Bernardo et al., 1974; White, 1984; Holmes & Lantz, 2001;
Patton, 2002; Doménech-Carbó, 2008; Sandu et al., 2012!.
There are noncovalent dyes as well as covalent probes
for protein detection ~Sun et al., 2004!. The dyes serving as
noncovalent probes are almost all anionic dyes. These dyes
can bind with positively charged protein residues, and so
pH is an important parameter. Upon binding to proteins,
the fluorescence intensity of the dyes may be enhanced or
quenched. The enhancement of a dyes’ fluorescence comes
mainly from a change in their microenvironment. Gener-
ally, these probe reagents are nonfluorescent in water, but
Cross-Section and Staining Techniques for Art 867
lem
~nm! Binding Media to be Detected
460 Proteins, peptides, and amino acids
525 Materials containing reactive amine
and alcohol groups ~mainly proteins!
625 Drying oils, fats
570 Proteins, shellac gums ~check cross
reactivity with materials containing
free amino groups, such as cationic
detergents!
590 Materials bearing amino groups
@NH2 # ~proteins!
525 Proteins and lipid droplets
518 Proteins
503–510 Lipids
610 Proteins
~535–750!
570 Proteins
~500–700!
highly fluorescent in apolar media. Given the three-
dimensional structure of proteins, these dyes can bind to
the hydrophobic region of a protein through noncovalent
binding, and their fluorescence yields are greatly enhanced.
Typical dyes of this type include naphthalene derivatives
~Horowitz & Bowman, 1987!, SYPRO dyes ~Steinberg et al.,
1996!, and Nile red ~Alba et al., 1996!.
Fluorescamine is a nonfluorescent compound until it
reacts with primary amine groups when it yields a blue-
white fluorescence. It is delivered in acetone because it is
easily hydrolyzed in water. Like FITC, it reacts better with
denatured aged proteins due to the greater availability of
primary amine groups ~De Bernardo et al., 1974!. Rinuy and
Gros ~1989! obtained good results on aged paint samples
but found it did not stain fresh protein layers. They found
that staining was better when fluorescamine was applied in
dioxane ~0.0026 M!, and it was particularly good for indicat-
ing the presence of egg white varnish. Since the unreacted
material is nonfluorescent, it is not necessary to remove the
excess. However, the blue-white fluorescence is similar, in
varying degrees, to the autofluorescence of lead white and of
proteinaceous binders. Wolbers and Landrey ~1987! suggest
the use of this reagent for materials that do not autofluoresce.
Fluoresceins are amine-reactive organic fluorophores
widely used to label proteins ~Nairn, 1962; Maeda et al.,
1969; Holmes & Lantz, 2001; Wang et al., 2010!. A variety of
fluorescein dyes are available for staining proteins and a
popular choice is fluorescein-5-isothiocyanate, known as
FITC. This dye forms covalent bonds with primary amines
in proteins ~Maeda et al., 1969!, or it binds with other
molecules, such as cellulose derivatives ~Laaser et al., 2011!,
868 Irina C.A. Sandu et al.
giving a bright yellow-green fluorescence. According to
Townsend, this stain is particularly effective on denatured
samples, as proteinaceous binders ~Rinuy & Gros, 1989!,
and on whole egg in particular ~Spring, 1991!. It has been
found to be quite sensitive and reliable on paint samples
even though it has a tendency to overstain the sample. A
major advantage is that its fluorescence has a distinctly
different hue from the blue-white autofluorescence of pro-
teins ~Rinuy & Gros, 1989!. Generally, FITC has relatively
highly absorption, high fluorescence quantum yield, and
aqueous solubility. Additionally, due to the fact that its
excitation maximum at 494 nm closely matches the 488 nm
spectral line of the argon-ion laser, FITC is the predominant
fluorophore for CLSM in conservation science applications.
However, FITC fluorophore has several major drawbacks
that include photobleaching ~its fluorescence rarely lasts
more than a few minutes under constant illumination! and
pH sensitivity ~its fluorescence depends on the pH of the
environment and significantly diminishes when pH value is
below 7!. This phenomenon also limits the sensitivity that
can be obtained using FITC. It has also been reported that
antibody conjugates prepared from FITC deteriorate over
time ~Vagnini et al., 2008; Cartechini et al., 2010!.
The fluorophore Rhodamine B is often used in biologi-
cal assays, being inexpensive and robust under a variety of
reaction conditions; can be covalently linked to bioactive mol-
ecules; and has suitable spectral properties in terms of absorp-
tion and fluorescence wavelength for applications in art
investigation field ~Arbeloa et al., 1991; Birtalan et al., 2011!.
Tetramethyl Rhodamine-Isothiocyanate ~TRITC!, like
LISSA, reacts with primary amines and alcohols so it can
stain starches and gums as well as proteins. It is dissolved in
dry acetone and gives a reddish fluorescence ~Wolbers,
1990, 2000!.
Lissamine Sulfonyl Chloride ~LISSA! has been used by
Wolbers ~1990!; it reacts with both primary amines and
alcohols; therefore, it is not as specific as some other fluoro-
chromes. A disadvantage is that it hydrolyses rapidly; there-
fore, it is important that the delivering solvent would be
dried ~Wolbers & Landrey, 1987!.
Nile Red is a dye used to stain proteinaceous materials
~Fowler & Greenspan, 1985; Greenspan et al., 1985!. The
disadvantage of Nile Red is its instability and the tendency
to precipitate in aqueous solutions, consequently making it
more difficult to use than commercial fluorescent stains.
DyLight ~488 nm! is a more stable and highly intense
fluorophore than FITC for labeling antibodies in immuno-
fluorescence techniques ~IFM!; its properties will be dis-
cussed with respect to the immunofluorescence fluorophores.
SYPRO stains ~SYPRO Red, SYPRO Orange, SYPRO
Ruby! ~Schäfer, 1997; Wolbers, 2000; Patton, 2002; Sandu
et al., 2009b, 2012! are noncovalent stains used in two- and
three-dimensional gels for electrophoresis in proteomics.
These stains could be an alternative to traditional staining
tests for mapping protein-based binders in cross sections
~Schäfer, 1997; Sandu et al., 2012!. This fluorescent protein
stain has several advantages ~Sandu et al., 2009b, 2012! that
make it useful for applications in conservation field: nano-
gram sensitivity ~lower detection limit than other protein
stains!; very high selectivity; straightforward staining proce-
dure ~fixation, incubation, no washing!; ready-to-use solu-
tion. The stain color is a bright yellow-orange ~Fig. 4! and
stains all proteinaceous materials, independently of their
biological origin ~mammalian or fish glues; egg, whole,
yolk, or glair; casein! or type of inert material/pigment with
which they are mixed in the paint layer ~Sandu et al., 2012!.
If the stained paint layer contains autofluorescent pigments/
dyes ~with a color similar to that of the stain, such as
Alizarin! there might be some interference/overlapping in
the staining results ~Sandu et al., 2012!.
F LUOR ESCENT S TAINING TESTS FOR F ATTY
AND L IPID M ATERIALS
Although it is widely mentioned and used as a stain for
proteins, Nile Red was also mentioned as an excellent stain
for detecting intracellular ~and particularly neutral! lipid
droplets by fluorescence microscopy ~Fowler & Greenspan,
1985; Greenspan et al., 1985!. Nile Red is almost nonfluores-
cent in water and other polar solvents but undergoes fluo-
rescence enhancement and large absorption and emission
blue shifts in nonpolar environments ~excitation/emission
maxima ; 552/636 nm in methanol!. A comparative study
with Oil Red O, used for lipid staining, was also described
~Jiney & Burgess, 2006!.
Nile Blue is generally used for histological staining
of biological preparations ~http://en.wikipedia.org/wiki/
Hystological_staining!. It highlights the distinction between
neutral lipids ~triglycerides, steroids, etc.!, which are stained
pink, and acid lipids ~fatty acids, phospholipids!, which are
stained blue ~Klinkner et al., 1997; Van Staveren et al.,
2001!. The absorption and emission maxima of Nile blue
are strongly dependent on the pH and on the solvents used
~Van Staveren et al., 2001!.
BODIPY威 dyes are normally used as stains for pep-
tides, phosphoproteins, neutral lipids, and as markers for
oils and other nonpolar liquids ~Johnson et al., 1991; Arbe-
loa et al., 1999; Cooper et al., 2010!. This dye group is
characterized by the combination of nonpolar structure and
a long-wavelength absorption and fluorescence. Flow cytom-
etry has shown that staining with BODIPY威 493/503 ~D-
3922! is more specific for cellular lipid droplets than staining
with Nile Red ~Gocze & Freeman, 1994!; therefore, its novel
application was considered for replicas of paint samples. A
preliminary test was done, and it seems that UV excitation
would be appropriate for visualizing BODIPY威 493/503
staining for oil-based paint layers, while the visible ~blue!
excitation is not as useful for displaying a specific stain for
oily material ~the green color of fluorescence is present both
in the ground layer and in the oil paint—Fig. 5!.
Immunofluorescence Microscopy
Biochemistry has developed specific protein labeling by using
immunological reagents ~specific antibodies! marked with a

Figure 4. Example of staining with SYPRO Ruby on cross sections of samples from two tempera paint replicas
containing different proteinaceous binders.
fluorescent dye that were recently reported in the identifica-
tion of paint binders ~Pinna et al., 2009; Cartechini et al.,
2010!. Jones ~1962! was the first to use the immunological
method in cultural heritage applications and specifically on
solutions of proteins extracted from the paint binder, but he
was not able to extract samples from aged paint media. Later,
Kockaert used the technique on cross sections from 15th
century paintings ~Kockaert et al., 1989!, but the results were
not particularly reliable. Afterward immunofluorescence
~IFM! on cross sections was reported to be successful for
recognizing casein, milk, and ovalbumin in paint layers ~Vag-
nini et al., 2008; Pinna et al., 2009!.
The immunological approach is based on the specific
interaction between the antigen ~protein present in the
sample! and its corresponding antibody ~immunoglobulin
protein! ~Doménech-Carbó, 2008; Vagnini et al., 2008; Sandu
et al., 2009b; Cartechini et al., 2010!. Extracted antibodies
can be monoclonal or polyclonal ~Wild, 2005!. The mono-
clonal type recognizes only one antigenic site ~epitope!
while the polyclonal antibodies are able to identify different
Cross-Section and Staining Techniques for Art 869
epitopes in a given protein. For samples from early paint-
ings in which protein aging/degradation caused a loss of
epitopes, polyclonal antibodies could increase the chances
to identify the target protein ~Pinna et al., 2009!.
Proteins, polysaccharides, and nucleic acids are effective
antigens; therefore, if an antibody is synthesized from a par-
ticular protein, it will have specific affinity for the amino acid
sequence in the protein. An antibody-antigen ~protein! com-
plex is formed and if the antibody is labeled with a fluoro-
phore, it will stain the protein. These antibodies could also be
labeled with a heavy metal, such as gold or silver, which would
make them visible under a scanning electron microscope
~Brad & Roth, 1984; Heginbotham et al., 2004!. Disadvan-
tages of this method are the short shelf life of fluorophores,
and the necessarily long exposure to aqueous solutions that
may dissolve soluble components of the paint samples. Since
antibodies are hydrophobic, there is also a possibility of bind-
ing with hydrophobic substances in the paint sample and
adsorption onto pigment particles ~Ramirez-Barat & de la
Vina, 2001!. In case of IFM on cross sections, the interference
870 Irina C.A. Sandu et al.
Figure 5. Example of BODIPY威 493/503 staining on a cross section from a paint replica.
of unspecific emissions from the samples is a major limita-
tion of this technique ~Pinna et al., 2009!.
New fluorophores, more resistant to bleaching phenom-
ena and with a higher fluorescence yield, were lately pro-
posed such as DyLight as alternative to FITC for labeling
antibodies specific to proteins. DyLight 488 ~Arslanoglu
et al., 2011!, in particular, has greater photostability than
traditional fluorescein-based dyes and has a lower sensitiv-
ity to the pH ~working range pH 4–9!. DyLight 488 is
available as a reactive labeling agent and as a conjugate of
secondary antibodies and biotin-binding proteins for use in
fluorescence microscopy, ELISA ~Enzyme-linked Immuno-
sorbent Assay!, and other biochemical assays ~e.g., flow
cytometry, Western blotting!. Figure 6 shows an example of
the positive use of the dye as a marker for secondary
antibodies in IFM tests on cross sections.
A combination of the stain Oil Red O with immunoflu-
orescence technique was reported for detecting and quanti-
fying lipids in biological products ~Koopman et al., 2001!,
but no applications of this stain have been found in the
conservation literature.
S TUDYING R ESTORATION TR EATMENTS
U SING F LUOR ESCENT D YES
Fluorescent dyes can be also used to monitor restoration
interventions, such as the cleaning of layers containing
protein-based materials ~egg white, isinglass, casein! or other
coatings. Wolbers ~Wolbers & Landrey, 1987; Wolbers, 1990,
2000! was the first to propose fluorescent stains ~such as
Rhodamine! for evaluating the performances of cleaning
methods. Laaser et al. ~2011! described the use of FITC to
show the migration of hydroxyl-propyl-cellulose for a wall-
paper consolidated with this cellulose derivative.
Figure 7 shows an innovative application of a fluores-
cent stain for proteins ~SYPRO Ruby! on cross sections of
samples taken from the same area of a paint replica, before
and after removal of the varnish layer using an enzyme-
based formulation.a
C ONCLUSIONS
Considerable advances in staining techniques have been made
since the early studies performed on cross sections of paint
samples, and the literature reports improvements and com-
bined applications of stains and immunological techniques
using fluorophores ~IFM!. Nevertheless, many other studies
can be done, considering that paint materials are intricate
mixtures with complex behavior and degradation mecha-
nisms. Most of the stains and fluorophores reported in con-
servation studies were borrowed from hystochemical or
molecular biology/proteomics applications, and therefore
they require specific testing on replicas of paint/polychrome
before validating the protocols on real samples. From the
reviewed literature it seems obvious that certain staining
agents act better on specific materials, such as Dylight-
labeled antibodies rather than the FITC-labeled antibodies
to detect protein-based binders ~casein, egg, fish, glue!; Amido
a
Work in progress in the framework of a Master’s thesis at DCR-FCT/UNL
in Portugal.

Figure 6. Positive IFM mapping and identification of ~a! ovalbumin on a cross section of a replica containing whole egg
in the upper yellow ochre layer ~egg tempera technique!; ~b! fish collagen used as binder for the ground layer. The
primary and secondary antibodies are shown.
Black for egg yolk, casein, and animal glue proteins against
Ponceau S ~that can yield misleading results in the presence
of calcium carbonate or gypsum due to the interference
caused by the Ca 2⫹ cation!; SYPRO Ruby for protein stain-
ing than other fluorescent stains ~LISSA, Nile Red, SYPRO
Orange!; Oil Red O for drying oils against Sudan Black.
It is a well-known fact that conservation laboratories,
and especially in private practice, do not have the financial
resources to be able to undertake laborious, expensive analy-
ses to identify materials and map paints ~especially those
which were added later through restoration, repainting/
overpainting, or attempts at forging works of art! that may
be crucial factors in decisions made by conservators-
restorers. Therefore, we selected a few case studies ~replicas
of paint composites! to illustrate the use of autofluores-
cence and induced fluorescence ~mainly using staining tests
specific to proteinaceous and oil-based paint materials! in
the cross-section mapping of both artificially and naturally
aged organic materials. Due to aging and the complex
nature of the matrix, samples from real works of art can
present difficulties in the interpretation of fluorescence
images. Furthermore, the influence of restoration interven-
tions that add new materials ~i.e., glues/resins in case of
facing and consolidation! to an original composition must
be also be considered in any extensive systematic study on
the use of the fluorescent stains.
We also propose some future directions for research
that could be further exploited for direct applications in
art conservation. These research areas include the comple-
Cross-Section and Staining Techniques for Art 871
mentary use of two different stains on the same cross
section/sample for the simultaneous identification of differ-
ent organic materials ~such as proteins and oils!; the char-
acterization of aged versus fresh materials through their
intrinsic and induced fluorescence; and the applicability of
cross-section observation and staining for assessing the
effectiveness of restoration treatments such as cleaning and
consolidation. This type of analytical information would be
of primary interest, especially for applications involving
valuable and unique works of art, for which sampling
always has to be minimal and as representative of the whole
layered structure as possible.
A CKNOWLEDGMENTS
This project was supported by Fundação para a Ciência e a
Tecnologia in Portugal through grant no. PEst-C/EQB/
LA0006/2011, and grant no. PTDC/EAT-EAT/116700/2010
and partially funded by CHARISMA - Cultural Heritage
Advanced Research Infrastructures: Synergy for a Multidis-
ciplinary Approach to Conservation/Restoration, GA No.
FP7-228330.
The authors would like to thank Catarina Pereira ~DCR-
FCT, Lisbon! for preparing cross sections and for staining
some samples and Marina Ginanni and Elena Prandi, restor-
ers at the Laboratorio Restauri della Soprintendenza Spe-
ciale per il Polo Museale Fiorentino ~Florence! and the
Director Dr. Magnolia Scudieri for providing historically
accurate replicas.
872 Irina C.A. Sandu et al.
Figure 7. Example of the application of the SYPRO Ruby stain for protein to assess the effectiveness of cleaning on egg
tempera paint.
R EFER ENCES
Alba, F.J., Bermudez, A., Bartolome, S. & Daban, J.R. ~1996!.
Detection of five nanograms of protein by two-minute Nile red
staining of unfixed SDS gels. BioTechniques 21, 625–626.
Aldrovandi, A. & Picollo, M. ~2001!. Metodi di documentazione
e indagini non-invasive sui dipinti. Il Prato, Collana i Talenti.
Appolonia, L. & Volpin, S. ~2001!. Le analisi di laboratorio appli-
cate ai beni artistici policromi. Padova, Italy: Il Prato.
Arbeloa, F.L., Arbeloa, T.L. & Arbeloa, I.L. ~1999!. Electronic
spectroscopy of pyrromethene 546. J Photochem Photobiol A
121, 177–182.
Arbeloa, F.L., Arbeloa, T.L., Estevez, M.J. & Arbeloa, I.L.
~1991!. Photophysics of rhodamines. Molecular structure and
solvent effects. J Phys Chem 95~6!, 2203–2208.
Arslanoglu, J., Zaleski, S. & Loike, J. ~2011!. An improved
method of protein localization in artworks through SERS
nanotag-complexed antibodies. Anal Bioanal Chem 399~9!,
2997–3010.
Banks, P.R. & Paquette, D.M. ~1995!. Comparison of three
common amine reactive fluorescent-probes used for conjuga-
tion to biomolecules by capillary zone electrophoresis. Biocon-
jug Chem 6~4!, 447–458.
Barton, H. ~2007!. Starch residues on museum artefacts: Implica-
tions for determining tool use. J Arch Sci 34~10!, 1752–1762.
Beaudoin, A. ~2004!. New technique for revealing latent finger-
prints on wet, porous surfaces: Oil Red O. J Forensic Identifica-
tion 54~4!, 413–421.
Becker, E. ~1989!. Fluorescence Microscopy. Germany: Wild Leitz
GmbH.
Birtalan, E., Rudat, B., Kolmel, D.K., Fritz, D., Vollrath,
S.B.L., Schepers, U. & Brase, S. ~2011!. Investigating rhoda-
mine B-labeled peptoids: Scopes and limitations of its applica-
tions. Peptide Sci 96~5!, 694–701.
Bousfield, B. ~1992!. Surface Preparation and Microscopy of Mate-
rials. New York: Wiley.
Brad, D. & Roth, J. ~1984!. ‘Golden blot’—Detection of poly-
clonal and monoclonal antibodies bound to antigens on nitro-
cellulose by Protein A—Gold complexes. Anal Biochem 142,
79–83.
Carlyle, L. ~2001!. The Artist’s Assistant. Oil Painting Instruction
Manuals and Handbooks in Britain 1800–1900 with Reference
to Selected Eighteenth-Century Sources. London: Archetype
Publications.
Cartechini, L., Vagnini, M., Palmieri, M., Pitzurra, L., Mello,
T., Mazurek, J. & Chiari, G. ~2010!. Immunodetection of
proteins in ancient paint media. Accounts Chem Res 43, 867–876.
Cooper, M.S., Hardin, W.R., Petersen, T.W. & Cattolico, R.A.
~2010!. Visualizing “green oil” in live algal cells. J Biosci Bioeng
109 ~2!, 198–201.

De Bernardo, S., Weigele, M., Toome, V., Manhart, K., Leimgru-
ber, W., Böhlen, P., Stein, S. & Udenfriend, S. ~1974!.
Studies on the reaction of fluorescamine with primary amines.
Arch Biochem Biophys 163, 390–399.
De La Rie, R. ~1982!. Fluorescence of paint and varnish layers,
Part I–II–III. Studies in Conservation 27~Pt I!, 1–7; 27~Pt II!,
65–69; 27~Pt III!, 102–108.
Derndarsky, M. & Ocklind, G. ~2001!. Some preliminary ob-
servations on subsurface damage on experimental and archae-
ological quartz tools using CLSM and dye. J Arch Sci 28,
1149–1158.
Derrick, M., Souza, L., Kieslich, T., Florsheim, H. & Stulik,
D. ~1994!. Embedding paint cross-section samples in polyester
resins: Problems and solutions. J Am Inst Conservation 33~3!,
227–245.
Deyl, Z., Mikšik, I. & Zicha, J. ~1999!. Multicomponent analysis
by off-line combination of synchronous fluorescence spectros-
copy and capillary electrophoresis of collagen glycation ad-
ducts. J Chromatogr A 836, 161–171.
Doménech-Carbó, M.T. ~2008!. Novel analytical methods for
characterizing binding media and protective coatings in art-
works. Anal Chim Acta 621, 109–139.
Dorge, V. & Carey Howlett, F. ~Eds.! ~1994!. Painted wood:
History and conservation. Proceedings of a symposium orga-
nized by the Wooden Artifacts Group of the American Institute for
Conservation of Historic and Artistic Works and the Foundation
of the AIC, Colonial Williamsburg Foundation, Williamsburg,
Virginia, November 11–14, 1994.
Feigl, F. & Anger, V. ~1966!. Spot-Tests in Organic Analysis, 7th ed.
New York: Elsevier.
Fowler, S.D. & Greenspan, P. ~1985!. Application of Nile Red, a
fluorescent hydrophobic probe, for the detection of neutral
lipid deposits in tissue sections: Comparison with Oil Red O. J
Histochem Cytochem 33~8!, 833–836.
Garrido, M. Del C. & Cabrera, J.M. ~1986!. Cross sections. In
PACT 13, 155–169.
Gay, M.C. ~1970!. Essais d’identification et de localisation des
liants par des colorations. In Annales du Laboratoire de Recher-
che des Musées de France, pp. 8–24.
Gay, M.C. ~1978!. Application of the staining method to cross-
sections in the study of the media of various Italian paintings
of the fourteenth and fifteenth centuries. In Conservation and
Restoration of Pictorial Art, Bromelle, B. & Smith, P. ~Eds.!,
pp. 78–83. London: Butterworths.
Gettens, R.J. & Stout, G.L. ~1966!. Painting Materials: A Short
Encyclopaedia. New York: Dover Publications.
Gocze, P.M. & Freeman, D.A. ~1994!. Factors underlying the
variability of lipid droplet fluorescence in MA-10 Leydig tumor
cells. Cytometry 17, 151–158.
Green, M.R., Pastewka, J.V. & Peacock, A.C. ~1973!. Differential
staining of phosphoproteins on polyacrylamide gels with a
cationic carbocyanine dye. Anal Biochem 56, 43–51.
Greenspan, P., Mayer, E.P. & Fowler, S.D. ~1985!. Nile Red—A
selective fluorescent stain for intracellular lipid droplets. J Cell
Biol 100, 965–973.
Heginbotham, A., Millay, V. & Quick, M. ~2004!. The use of
immunofluorescence microscopy ~IFM! and enzyme-linked im-
munosorbent assay ~ELISA! as complementary techniques for
protein identification in artists’ materials. J Am Inst Conserva-
tion 45~2!, 89–105.
Holmes, K.L. & Lantz, L.M. ~2001!. Protein labeling with fluores-
cent probes. Methods Cell Biol 63, 185–204.
Cross-Section and Staining Techniques for Art 873
Horowitz, P.M. & Bowman, S. ~1987!. Ion-enhanced fluorescence
staining of sodium dodecyl sulfate-polyacrylamide gels using
bis~8-p-toluidino-1-naphthalenesulfonate!. Anal Biochem 165,
430–434.
James, J. & Tas, J. ~1984!. Histochemical protein staining methods.
In Microscopy Handbooks Vol. 4. Oxford, UK: Royal Microscop-
ical Society, Oxford University Press.
Jiney, J. & Burgess, K. ~2006!. Benzophenoxazine-based fluores-
cent dyes for labeling biomolecules. Tetrahedron 62, 11021–
11037.
Johnson, I.D., Kang, H.C. & Haugland, R.P. ~1991!. Fluorescent
membrane probes incorporating dipyrrometheneboron difluo-
ride fluorophores. Anal Biochem 198, 228–237.
Johnson, M. & Packard, E. ~1971!. Methods used for identifica-
tion of binding media in Italian paintings. Studies in Conserva-
tion 16, 145–164.
Jones, P.L. ~1962!. Some observations on methods for identifying
proteins in paint media. Stud Conserv 7, 10–16.
Karpowicz, A. ~1981!. Ageing and deterioration of proteinaceous
media. Stud Conserv 26, 153–160.
Kay, R.E., Walwick, E.R. & Gifford, C.K. ~1964!. Spectral changes
in a cationic dye due to interaction with macromolecules. I.
Behaviour of dye alone in solution and the effect of added
macromolecules. J Phys Chem 68~7!, 1896–1916.
Khandekar, N. ~2003!. Preparation of cross-sections from easel-
paintings. Stud Conserv 48~Suppl 1!, 52–64.
Klinkner, A.M., Bugelski, P.J., Robbie Waites, C., Louden, C.,
Hart, T.K. & Kerns, W.D. ~1997!. A novel technique for
mapping the lipid composition of atherosclerotic fatty streaks
by en face fluorescence microscopy. J Histochem Cytochem
45~5!, 743–753.
Kockaert, L, Gausset, P. & Dubi-Rucquory, M. ~1989!. Detec-
tion of ovalbumin in paint media by immunofluorescence.
Stud Conserv 34, 183–188.
Koopman, R., Schaart, G. & Hesselink, M.K.C. ~2001!. Optimi-
sation of Oil Red O staining permits combination with immu-
nofluorescence and automated quantification of lipids.
Histochem Cell Biol 116, 63–68.
Kühn, H. ~1986!. Conservation and Restoration of Works of Art and
Antiquities, vol 1, pp. 157–167. London: Butterworths.
Laaser, T., Stoppa, K. & Krekel, C. ~2011!. Making consolidation
visible: Examination of hydroxy propylcellulose migration dur-
ing consolidation of a painted wallpaper using a fluorescent-
labeled consolidant. In The Sticking Point: Adhesives and
Consolidants in Paintings Conservation Icon Paintings Group
Conference, May 6, 2011. London: National Portrait Gallery.
Maeda, H., Ishida, N., Kawauchi, H. & Tuzimura, K. ~1969!.
Reaction of FITC with proteins and amino acids. J Biochem
65~5!, 777–783.
Martin, E. ~1975!. Note sur l’identification des proteines dans les
liants de peinture. In Annales du Laboratoire de Recherche des
Musées de France, pp. 57–60.
Martin, E. ~1977!. Some improvements in the techniques of
analysis of paint media. Stud Conserv 22, 63–67.
Martin, E. ~1978!. Application des tests sur coupes minces á
1’identification des emulsions dans les liants de peinture. In
Annales du Laboratoire de Recherche des Musées de France,
pp. 23–29.
Martin, J.S. ~1994!. Microscopic examination and analysis of the
structure and composition of paint and varnish layers. In
Painted Wood: History and Conservation, Proceedings of the
Symposium Organized by the Wooden Artifacts Group of the
874 Irina C.A. Sandu et al.
American Institute for Conservation of Historic and Artistic Works.
pp. 64–75. Los Angeles, CA: The Getty Conservation Institute.
Masschelein Kleiner, L. ~1992!. Liants, vernis et adhesifs anciens.
Brussels, Belgium: Bruxelles Institut Royal du patrimoine artis-
tique KIK-IRPA.
Matteini, M. & Moles, A. ~1984!. Scienza e restauro. Metodi di
indagine. Florence, Italy: Nardini Editore.
Matteini, M. & Moles, A. ~2002!. La Chimica nel Restauro. I
materiali dell’arte pittorica. Florence, Italy: Nardini Editore.
Matteini, M., Moles, A. & Tosini, I. ~1981!. Topochemical reac-
tions for the recognition of oil media in paint fragments. In
ICOM-CC Triennial Meeting Proceedings, Ottawa, Canada.
Messinger, J.M. ~1992!. Ultraviolet-fluorescence microscopy of
paint cross-sections. J Am Inst Conservation 31~3!, 267–274.
Mills, J.S. & White, R. ~1979!. Analysis of paint media. National
Gallery Technical Bulletin 3, 66–67.
Mills, J.S. & White, R. ~1994!. The Organic Chemistry of Museum
Objects, 2nd ed. London: Butterworth-Heinemann.
Moles, A., Matteini, M. & Tosini, I. ~1982!. Le tecniche microanal-
itiche condotte su sezioni. In Metodo e Scienza—Operativitá e
ricerca nel restauro, pp. 271–273. Florence, Italy: Sansoni Editore.
Nairn, R.C. ~Ed.! ~1962!. Fluorescent Protein Tracing. Edinburgh:
Livingstone.
Osmond, G. ~1993!. Accelerated deterioration of artists’ oil paints:
An assessment involving ultraviolet fluorescence spectroscopy.
In 10th Triennial Meeting ICOM Committee for Conservation,
preprints, Bridgland, J. ~Ed.!. London: James and James.
Patton, W.F. ~2002!. Detection technologies in proteome analysis.
J Chromatogr B 771, 3–31.
Petraco, N. & Kubic, T. ~2004!. Color Atlas and Manual of
Microscopy for Criminalists, Chemists and Conservators. Boca
Raton, London, New York, Washington, D.C.: CRC Press.
Petty, H.R. ~2007!. Fluorescence microscopy: Established and
emerging methods, experimental strategies, and applications in
immunology. Microsc Res Tech 70, 687–709.
Phenix, A. ~1997!. The composition and chemistry of eggs and
egg tempera. In Early Italian Paintings: Techniques and Analysis:
Symposium, Bakkenist, T., Hoppenbrouwers, R. & Dubois, H.
~Eds.!, pp. 11–12. Maastricht, The Netherlands: Limburg Con-
servation Institute.
Pilc, J. & White, R. ~1995!. The application of FTIR-microscopy
to the analysis of paint binders in easel paintings. National
Gallery Technical Bulletin 16, 73–84.
Pinna, D., Galeotti, M. & Mazzeo, R. ~Eds.!. ~2009!. Scientific
Examination for the Investigation of Paintings: A Handbook for
Conservators-Restorers. Florence, Italy: Centro Di.
Plesters, J. ~1956!. Cross-sections and chemical analysis of paint
samples. Stud Conserv 2~3!, 110–157.
Ploem, J.S. & Tanke, H.J. ~1987!. Introduction to Fluorescence
Microscopy. Oxford, UK: Oxford University Press, Royal Micro-
scopical Society.
Ramirez-Barat, B. & de la Vina, S. ~2001!. Characterization of
proteins in paint media by immunofluorescence. A note on
methodological aspects. Stud Conserv 46~4!, 282–288.
Rinuy, A. & Gros, L. ~1989!. Liants dans les peintures anciennes:
Methodes d’identification et Etude du vieillissement. Zeitschrift
für Kunstechnologie und Konservierung, Heft 1, Jahrgang, 3, 9–39.
Rosi, F., Federici, A., Brunetti, B.G., Sgamellotti, A., Cle-
menti, S. & Miliani, C. ~2011!. Multivariate chemical map-
ping of pigments and binders in easel painting cross-sections
by micro IR reflection spectroscopy. Anal Bioanal Chem 399~9!,
3133–3145.
Sandu, I.C.A., Bracci, S., Lobefaro, M. & Sandu, I. ~2010!.
Integrated methodology for the evaluation of cleaning effective-
ness in two Russian icons ~16th–17th centuries!. Microsc Res
Tech 73, 752–760.
Sandu, I.C.A., Bracci, S. & Sandu, I. ~2006!. Instrumental analy-
ses used in the authentication of old paintings. I. Comparison
between two icons of XIXth century. Rev de Chim ~Bucharest!
57~7!, 796–802.
Sandu, I.C.A., Bracci, S., Sandu, I. & Lobefaro, M. ~2009a!.
Integrated analytical study for the authentication of five
Russian icons ~16th–17th centuries!. Microsc Res Tech 72,
755–765.
Sandu, I.C.A., Luca, C., Sandu, I., Vasilache, V. & Hayashi, M.
~2008!. Authentication of ancient easel-paintings through ma-
terials identification from polychrome layers. III. Cross-section
analysis and staining tests. Rev de Chim ~Bucharest! 59~8!,
785–793.
Sandu, I.C.A., Roque, A.C.A, Kuckova, S., Schäfer, S. & Car-
reira, R. ~2009b!. The biochemistry and artistic studies: A
novel integrated approach to the identification of organic bind-
ers in polychrome artifacts, in the first issue of ECR. Estudos de
Conservação e Restauro, pp. 39–56. Oporto, Portugal: CITAR
Escola das Artes, Universidade Católica Portuguesa.
Sandu, I.C.A., Roque, A.C.A., Matteini, P., Schäfer, S., Agati,
G., Ribeiro Correia, C. & Fortio Fernandes Pacheco, J.
~2012!. Fluorescence recognition of proteinaceous binders in
works of art by a novel integrated system of investigation.
Microsc Res Tech 75~3!, 316–324.
Sandu, I.C.A, Sandu, I. & Luca, C. ~2005!. Modern Aspects Con-
cerning the Conservation of the Cultural Heritage. Vol. II. Authen-
tication and Determination of the Preservation State of Ancient
Paintings. Iasi, Romania: Performantica.
Schäfer, S. ~1997!. Fluorescent staining techniques for the charac-
terization of binding media within paint cross sections and
digital image processing for the quantification of staining re-
sults. In Postprints of the Symposium on Early Italian Painting
Techniques and Analysis, Bakkenist, T., Hoppenbrouwers, R. &
Dubois, H. ~Eds.!. Maastricht, The Netherlands: Limburg Con-
servation Institute.
Sciutto, G., Dolci, L.S., Buragina, A., Prati, S., Guardigli, M.,
Mazzeo, R. & Roda, A. ~2011!. Development of a multiplexed
chemiluminescent immunochemical imaging technique for the
simultaneous localization of different proteins in painting mi-
cro cross-sections. Anal Bioanal Chem 399, 2889–2897.
Siegel, A.B.G. ~1993!. Computer enhanced UV fluorescence micros-
copy on aged artists’ materials. Art Conservation Training
Conference, Buffalo, NY.
Slansky, B. ~1956!. Technika v malirske tvorbe ~Technique in
Painting Creation. Classical book about painting and restoring
materials. Pigmentation, thinners, grounds of paintings and
fresco!. Prague: Paseka.
Spring, K.R. & Davidson, M.W. ~2008!. Introduction to fluores-
cence microscopy. Nikon MicroscopyU. Available at http://
www.microscopyu.com/articles/fluorescence/fluorescenceintro.
html ~retrieved 2008-09-28!.
Spring, M. ~1991!. A study of staining techniques for the identi-
fication of proteinaceous paint media. Unpublished bach-
elor thesis. London: Cambridge University, Hamilton Kerr
Institute.
Steinberg, T.H., Haugland, R.P. & Singer, V. ~1996!. Applica-
tions of SYPRO Orange and SYPRO Red protein gel stains.
Anal Biochem 239~2!, 238–245.

Sun, C., Yang, J., Li, L., Wu, X., Liu, Y. & Liu, S. ~2004!. Advances
in the study of luminescence probes for proteins, J Chromatogr
B 803, 173–190.
Talbot, R.R. ~1982!. The fluorescent antibody technique in the
identification of proteinaceous materials. In Third Annual Con-
ference of Art Conservation Training Programs, Queens Univer-
sity, Kingston, Ontario.
Tsang, J. & Cunningham, R. ~1991!. Some improvements in the
study of cross-sections. J Am Inst Conserv 30, 163–177.
Udenfriend, S., Stein, S., Böhlen, P., Dairman, W., Leimgru-
ber, W. & Weigele, M. ~1972!. Fluorescamine: A reagent for
assay of amino acids, peptides, proteins, and primary amines in
the picomole range. Science 178, 871–872.
Vagnini, N., Pitzurra, L., Cartechini, L., Miliani, C., Bru-
netti, B.G. & Sgamelotti, A. ~2008!. Identification of pro-
teins in painting cross-sections by immunofluorescence
microscopy. Anal Bioanal Chem 392, 57–64.
Van Staveren, H.J., Speelman, O.C., Witjes, M.J., Cincotta, L.
& Star, W.M. ~2001!. Fluorescence imaging and spectroscopy
of ethyl Nile blue A in animal models of ~pre!malignancies.
Photochem Photobiol 73~1!, 32–38.
Wachowiak, M.J., Jr. ~2004!. Efficient new methods for embed-
ding paint and varnish samples for microscopy. J Am Inst
Conserv 43~3!, 205–226.
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Cross-Section and Staining Techniques for Art 875
Waentig, F. ~1993!. Gießharzsysteme zum Einbetten von Proben.
Restauro 3, 195–199.
Wang, F., Tan, W.B., Zhang, Y., Fan, X. & Wang, M.Q. ~2006!.
Luminescent nanomaterials for biological labeling. Nanotechnol-
ogy 17, R1.
Wang, F., Tan, W.B., Zhang, Y., Fan, X. & Wang, M. ~2010!.
Luminescent nanomaterials for biological labeling. Nanotechnol-
ogy 17~1!, R1–R13.
White, R. ~1984!. The characterisation of proteinaceous binders
in art objects. National Gallery Technical Bulletin 8, 5–14.
Wild, D. ~2005!. The Immunoassay Book, 3rd ed. Amsterdam, The
Netherlands: Elsevier.
Wolbers, R.C. ~2000!. Cleaning Painted Surfaces. Aqueous Meth-
ods. London: Archetype Publications.
Wolbers, R.C. ~1990!. Microscopy—Interpreting Stains (Notes for
workshop on new methods in the cleaning of paintings). Marina
del Rey, CA: Getty Conservation Institute.
Wolbers, R.C. & Landrey, G. ~1987!. The use of direct reactive
fluorescent dyes for the characterization of binding media in
cross sectional examinations. In Preprints of 15th Annual Meet-
ing of American Institute for Conservation, Washington, D.C.,
pp. 168–202.