Optics Communications 273 (2007) 219–225

www.elsevier.com/locate/optcom

Remote detection of laser-induced autofluorescence on pure cultures

of fungal and bacterial strains and their analysis with
multivariate techniques
a,*
Valentina Raimondi , Lorenzo Palombi a, Giovanna Cecchi a, David Lognoli a,
Massimo Trambusti a, Ioana Gomoiu b
a
National Research Council, Institute of Applied Physics ‘‘Nello Carrara’’ (CNR-IFAC), Via Madonna del Piano, 10, I-50019 Sesto Fiorentino,
Firenze, Italy
b
Institute of Biology, National Arts University of Bucharest, Splaiul Independentei, 296, R-77700 Bucharest, Romania

Received 5 July 2006; received in revised form 25 October 2006; accepted 7 December 2006

Abstract

Remotely sensed laser-induced autofluorescence spectra of pure cultures of fungal strains (Aureobasidium pullulans, Verticillium sp.)
and of bacterial strains (Bacillus sp., Pseudomonas sp.) are presented. The strains were isolated from samples collected in a Roman
archaeological site (Tropaeum Traiani) near Constanta, Romania. The fluorescence spectra were detected in vivo from a distance of
25 m in the outdoor, using a high spectral resolution fluorescence LIDAR featuring a UV laser (XeCl@308 nm) as an excitation source.
All the examined strains, except for the A. pullulans, showed fluorescence features such to allow their characterisation by processing data
with multivariate techniques. Both Principal Component Analysis and Cluster Analysis were applied to the data set and compared to
discriminate between the examined strains.
Results demonstrate the feasibility of fluorescence-based detection and characterisation of fungi and bacteria in the outdoor with a
high spectral resolution fluorescence LIDAR. In addition, they show that the proposed processing methods offer a means to discriminate
between the fluorescence features due to the investigated samples and that of a fluorescence background of a known spectral shape, as
that of the culture medium. This can be exploited for the remote fluorescence mapping of heterotrophic organisms on stone surfaces
when the latter show a typical broad fluorescence band.
Ó 2006 Elsevier B.V. All rights reserved.

1. Introduction able treatments for the eradication. Although these micro-

Biodeterioration is a typical process that affects monu-
ments in the outdoor: its monitoring, treatment and timely
prevention is thus essential for the conservation of the cul-
tural heritage. These actions, however, require the preli-
minary identification of these microorganisms, the
mapping of the contaminated areas and the choice of suit-
*
Corresponding author. Tel.: +39 055 5226379; fax: +39 055 5226201.
E-mail addresses: v.raimondi@ifac.cnr.it (V. Raimondi), l.palombi@
ifac.cnr.it (L. Palombi), g.cecchi@ifac.cnr.it (G. Cecchi), d.lognoli@
ifac.cnr.it (D. Lognoli), m.trambusti@ifac.cnr.it (M. Trambusti), ioana.
gomoiu@ibiol.ro (I. Gomoiu).
organisms can be thoroughly studied in the laboratory
[1,2,4], their effects and distribution can be only investi-
gated by the direct observation on the monument. Actu-
ally, the laboratory analysis first requires the collection of
samples, which are necessarily in a limited number, the pro-
tection of samples from contamination during handling,
transportation and cultivation in the laboratory. In addi-
tion, cultivation methods usually require synthetic nutrient
media having a chemical composition different from that of
the natural substrate with the consequence that not all
microorganisms present in situ can be isolated in the labo-
ratory. On the other hand, most in situ techniques [1,3,5,6]
presently available for the investigation on monuments are

0030-4018/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.optcom.2006.12.013
220 V. Raimondi et al. / Optics Communications 273 (2007) 219–225
time consuming or cannot be easily applied to extended
areas.
In this context remote sensing techniques for monitoring
biodeteriogens directly on monuments can offer several
advantages, especially as far as an overall evaluation of
biodeteriogens distribution over extended surfaces is con-
cerned. They can be exploited also for the individuation
of the most suitable sampling areas and for the in vivo
investigation of the biodeterioration process.
Fluorescence LIDAR remote sensing [7] makes it possi-
ble to apply the Laser-Induced Fluorescence (LIF) tech-
nique directly on monuments in the outdoor without the
collection of samples or the use of scaffolds. This technique
has already been applied to the diagnostics of the surfaces
of historical buildings [8], especially for the characterisa-
tion of lithotypes and for the detection and characterisa-
tion of photoautotrophic biodeteriogens [9,10]. However,
fluorescence of heterotrophic microorganisms has not yet
been investigated with the LIDAR remote sensing tech-
nique, especially as for their differentiation among species
and strains. Some data are available about LIDAR moni-
toring of fungi-affected insulator surfaces [11,12], while a
recent study presents the technique for the differentiation
of fungi in the laboratory with optical-fibre coupled instru-
mentation [13].
The present paper thus extends the results obtained on
fungal strains in the laboratory to the outdoor operation
with remote sensing instrumentation. In addition, it dem-
onstrates the feasibility of the remote detection of bacterial
strains and the use of multivariate analysis techniques,
both Principal Component Analysis and Cluster Analysis,
for their discrimination.
2. Experimental
The measurements here presented were conducted in
April 2004 within the frame of the EU-funded Culture
2000 project. The measurements were done in the outdoor,
in full sunlight, on pure cultures of fungal and bacterial
strains by using a high spectral resolution fluorescence
LIDAR, the FLIDAR, deployed at a distance of about
20 m from the sample. Data were then analysed by using
Table 1
Main characteristics of the samples
Label Sample description
Fungi F-blank Nutrient medium
F1 Black crusts
F2 White powdery
Bacteria B-blank Nutrient medium
B1-a Black crusts
B1-b Black crusts
B2-a Black crusts
B2-b Black crusts
a
Most frequently found genera in the sample. The two strains of Bacillus sp. and Pseudomonas sp. can either belong to different species of the same
genera or be different strains of the same species.
b
Units Forming Colonies, belonging to different genera of bacteria or fungi, found in the sample.
both Principal Component Analysis (PCA) and Cluster
Analysis (CA) techniques. The following subsections give
a description of the examined samples, of the instrumenta-
tion and of the data processing method used to achieve the
results presented.
2.1. Materials and methods
The fungal and bacterial strains (Table 1) were collected
in a Roman archaeological site, Tropaeum Traiani near
Constanta, Romania, during a sampling campaign carried
out within the frame of the same project.
The monument areas supposed to be contaminated by
microorganisms had two distinct morphologies: black
crusts and white powder. These types of morphologies
are common on deteriorated stones. The first one is in solid
state, while the second one is powdery. Because of its mor-
phology, the latter can be observed only before the surface
is washed by rain or in hidden areas. Black crusts are the
result of a multi-step colonization of the stone, while white
powder is a mixture of minerals, hyphae and fungal spores.
The collected samples taken under sterile conditions
were vigorously agitated in 10-min vortexing and then
diluted (1/10). Duplicate and triplicate 1-ml volumes of
appropriate dilutions were inoculated onto the culture
medium for bacteria (sodium acetate, yeast extract and
bacteriological peptone) and onto DPA (dextrose-pota-
toes-agar) for fungi. After 48 h and six days of incubation
at 28 °C for bacteria and for fungi, respectively, Units
Forming Colonies (UFC) were counted and pure culture
were isolated. The bacteria were identified in the laboratory
according to the morphology of colonies and of the cul-
tures, Gram staining and biochemical features. The fungi
were identified according to their cultural characteristics
and to the morphology of their hyphae and spores.
2.2. Instrumentation
The fluorescence measurements were carried out with a
high spectral resolution fluorescence LIDAR (FLIDAR-3),
developed at CNR-IFAC and operative aboard a small van
used as a mobile laboratory in the field.
Genusa UFC/sampleb
– –
Aureobasidium pullulans 1:2  103
Verticillium sp. 1:8  104
– –
Bacillus sp.1 1:0  102
Bacillus sp.2 1:6  104
Pseudomonas sp.1 1:5  104
Pseudomonas sp.2 1:3  105
 V. Raimondi et al. / Optics Communications 273 (2007) 219–225
The FLIDAR-3 features an excimer laser as excitation
source (XeCl@308 nm; typical pulse energy: 30 mJ; pulse
duration: 10 ns; repetition rate: 1–10 Hz). An excimer-
pumped dye laser (Coumarin@480 nm; 10% efficiency)
can be selected as an alternative excitation source. The
backscattered signal is collected with a 25-cm diameter
1-m focal length Newtonian telescope. An optical-fibre
bundle conveys the signal from the telescope image plane
to the input slit of a 275-mm focal length spectrometer
coupled to an intensified, gateable 512-photodiode array
detector. A set of selectable optical long-pass filters,
placed in front of the fibre bundle input, are used to
reject the laser wavelength and higher orders from the
collected signal. Control electronics, data acquisition
and data storage are controlled via a personal computer.
The system allows to acquire spectra in full sunlight
since the detector is gateable down to 100 ns and the
acquisition routines subtract from the active spectrum
the ambient light background acquiring it immediately
after each laser pulse. A detailed description of the sys-
tem is given in Ref. [14].
2.3. Experimental set-up
The measurements on the pure cultures isolated from
the collected samples were carried out in the outdoor in
full sunlight to reproduce as much as possible the exper-
imental conditions that can be found in the field. The
Petri dishes containing the bacterial and fungal pure cul-
tures were placed on a sample holder covered with a
non-fluorescent fabric. The sample holder was placed at
a 25-m distance from the sensor. At this distance the
area detected by the sensor on the sample was about
2 cm in diameter. The Petri dish containing the samples
was positioned so as to maximise the quantity of fun-
gal/bacterial units detected by the sensor. The van posi-
tion was made steady by a set of stabilizer jacks to avoid
unwanted shifts of the detected area during the
measurement.
Fluorescence spectra were acquired with the 308-nm
excitation wavelength at a repetition rate of 2 Hz. The laser
fluence per pulse at the target was less than 0.5 mJ/cm2. A
set of two different fluorescence spectra was acquired for
each measurement: the first spectrum ranges from 300 nm
to 600 nm and the second one from 500 nm to 800 nm with
a spectral resolution better than 2.4 nm. Each fluorescence
spectrum was the sum of 50 single acquisitions on the sam-
ple to improve the signal-to-noise ratio.
The areas where the measurements were taken were
always selected so as to include as much as possible fungi
or bacteria. Since the area measured on the sample was
about 2 cm in diameter and the sample distribution inside
the Petri dishes was not always homogeneous, the mea-
surements taken on the same sample, but on different
spots, could differ slightly. To take into account this
effect, different measurements were taken on different
spots of the same sample to evaluate possible differences.
221
Although the intensity of the fluorescence signal slightly
varies from one measurement to the other, the spectral
shape is almost unchanged: the correlation coefficients
among spectra taken on the same sample, but acquired
on different spots, vary between 0.999 and 0.987, depend-
ing on the sample.
Fluorescence spectra of the culture medium were also
acquired. The fluorescence contribution of the culture med-
ium is not negligible: however, as it will be explained in the
following, multivariate statistical techniques can be used to
point out differences in the spectral shape that are only due
to the fungi and bacteria cultures.
2.4. Data elaboration
The fluorescence spectra were corrected for the spectral
response of the instrumentation. Each set of two spectra,
the first spectrum ranging from 300 nm to 600 nm and
the second one from 500 nm to 800 nm, were merged and
then normalised to the Standard Deviation (STD) of the
merged spectrum. The normalisation procedure is usually
applied in remote sensing applications where absolute mea-
surements are difficult to perform. In general, with respect
to the normalisation to the maximum, the STD normalisa-
tion shows the advantage to take into account the features
of the whole spectrum and thus to be less sensitive to spec-
trum noise.
The normalised spectra were processed with the Princi-
pal Component Analysis (PCA) technique to analyse the
differences in the fluorescence spectral shapes and also with
the aim to separate the contribution of the culture medium
from fungal or bacterial strain fluorescence. The PCA is a
statistical multivariate technique that can be used to point
out similarities and differences in a set of spectra depending
on a high number p of variables. It consists in operating a
roto-translation of the coordinates system to maximize the
variance of a linear combination of the p variables: the
coordinates describing each spectrum in the new space
are called ‘scores’, while the orthonormal vectors spanning
the new space are called Principal Components (PCs). The
PC scores can be plotted against each other to reveal ‘clus-
tering’ features due to the spectral similarities of the spectra
[15].
Cluster Analysis (CA) was also applied to the data and
results were compared with those obtained with the PCA
technique. CA is an unsupervised classification method
used to arrange a set of data into clusters with the aim to
establish a set of clusters such that all data within a cluster
are more similar to each other than they are to data in
other clusters. In particular, data were processed with a
hierarchical clustering, using the Euclidean distance as an
index of dissimilarity between two observations and the
single linkage as linkage method, where in the latter the dis-
tance between two generic clusters A and B is the minimum
distance between a point in cluster A and a point in cluster
B [15]. The single linkage method is also called the ‘nearest
neighbour’ method.
222 V. Raimondi et al. / Optics Communications 273 (2007) 219–225

3. Results and discussion
The set of fluorescence spectra taken on the fungal and
bacterial strains are shown in Fig. 1. For each strain several
measurements were acquired in different spots of the same
sample. All the spectra show an intense fluorescence band
around 450 nm. The fluorescence spectra of the culture
media are reported in the graphs and labelled as F-blank
and B-blank for the fungal and the bacterial strains, respec-
tively. All the samples show a broad fluorescence band with
a maximum at about 450 nm and similar shapes, except for
a strain of Bacillus sp. (B1-b) that has quite a different spec-
tral shape with respect to the blank and to the other bacte-
rial strains. On the other hand, the only sample showing a
remarkable enhancement of the fluorescence intensity with
respect to the blank is a strain of Bacillus sp. (B1-a).
The STD-normalised spectra are shown in Fig. 2. For
the sake of clarity, the graph reports only one spectrum
for each sample: the spectrum shown in the graph is
obtained by operating a mean over the measurements car-
ried out on the same sample.
From Fig. 2a, it can be inferred that the Aureobasidium
pullulans sample (F1 in the figure) is very similar to the cor-
responding culture medium, while the other fungi sample
(Verticillium sp., F2 in the figure) shows a different spectral
shape from the culture medium (F-blank) because of a rel-
ative enhancement of the fluorescence in the red spectral
region. In the insertion a close-up shows a section of F1,
F-blank and the F2 curves. The lack of additional fluores-
cence features of the F1 sample with respect to the culture
medium ones is consistent with the fact that Aureobasidium
pullulans contains a black pigment that has the role of pro-

11
x 10
F1
14 F1 3.5 F2
F2 F-blank
F-blank 3
12 1
0.95
0.9
2.5 0.85
10
Intensity (a.u.)

0.8
Intensity (a.u.)

0.75
2 0.7
8 0.65
0.6
0.55
1.5
0.5
6 520 530 540 550 560 570 580

1
4

0.5
2

350 400 450 500 550 600 650 700

0
350 400 450 500 550 600 650 700 Wavelength (nm)
Wavelength (nm)
4
12 B1-a
x 10
B1-b
3.5
B1-a B2-a
5 B1-b B2-b
B2-a 3 B-blank
B2-b
B-blank
2.5
Intensity (a.u.)

2
Intensity (a.u.)

3
1.5

2 1

0.5
1
0
350 400 450 500 550 600 650 700
Wavelength (nm)
0
350 400 450 500 550 600 650 700
Wavelength (nm) Fig. 2. STD-normalized fluorescence spectra of: (a) fungal strains and
corresponding blank (the insertion shows a close-up of a section of the
Fig. 1. Fluorescence spectra taken on: (a) fungal strains and nutrient curves that allows to see all the three curves), and (b) bacterial strains and
medium (F-blank) and (b) bacterial strains and nutrient medium (B- corresponding blank. The spectrum shown for each sample is the mean
blank). spectrum of the spectra obtained on different spots of the same sample.
 V. Raimondi et al. / Optics Communications 273 (2007) 219–225
tecting the hyphae from UV radiation and the presence of
this pigment can thus inhibit the laser penetration into the
fungus.
Fig. 2b shows the normalized fluorescence spectra
obtained on the bacterial strains: a strain (B1-b in the fig-
ure) of Bacillus sp. shows a clearly different spectral shape
from the blank as well as from the other samples, including
the other strain (B1-a in the figure) of Bacillus sp. The fluo-
rescence spectrum is actually enhanced in the red with
respect to the other fluorescence spectra, as it could already
be inferred from Fig. 1b. All the other samples, although
featuring a spectral shape different from the blank, are
more similar to each other as far as their fluorescence fea-
tures are concerned.
The normalised spectra were analysed with the PCA
technique to evaluate the differences in the shapes of the
fluorescence spectra and also with the aim of separating
the fluorescence contribution of the culture medium from
that of the fungal or bacterial strains. The PCs that were
found more meaningful for data interpretation were the
first two PCs: the first PC (PC1), the second one (PC2)
and the third one account for, respectively, the 88.3%,
7.9% and the 2.2% of the total variance of the data. In
the whole, the first two PCs thus account for the 96.2%
of the total variance of the fluorescence spectra with respect
to the average spectrum. The PC1 and PC2 loading vectors
obtained applying the PCA technique to the complete set
of data are shown in Fig. 3. The shape of the PC1 loading
vector can be related to an enhancement in the red spectral
region with respect to the average spectral behaviour so
that a high score of this component must be related to an
enhancement of the fluorescence spectrum in this spectral
region. The PC2 loading vector can be associated with an
enhancement of the fluorescence contribution around the
maximum of the band (about 450 nm) with respect to the
0.08
0.06
0.04
0.02
0
0.02
0.04
0.06
0.08
0.1
350 400 450 500 550 600 650
Wavelength (nm)
Fig. 3. The loading vectors of the two first principal components, PC1 and
PC2, obtained applying the PCA to the complete set of data.
223
other fluorescence contributions centred around 400 nm
and 550 nm.
Fig. 4 displays the scores of the two first principal com-
ponents plotted against each other (PC2 score versus PC1
score). In general, these results confirm the inferences
drawn on the basis of the comparison among the spectral
shapes of the normalized fluorescence spectra (Fig. 2) and
further point out the differences between groups of data:
all the data referring to the F2 sample are clearly separated
from the data referring to the culture medium (F-blank)
and to the other fungal strain; the B1-a and B1-b bacterial
strains are well differentiated from all other samples and
the culture medium (B-blank). In particular, this graph
underlines the possibility of distinguishing between fungi
and bacteria as biodeteriogens, at least for the strains inves-
tigated, since the two classes of samples are separated in the
graph and overlapping occurs only with the nutrient media
of the opposite class. The same results were obtained with
the data normalised to the maximum, as expected in this
case where spectra are quite intense and the signal-to-noise
ratio is high.
For the sake of clarity the data reported in Fig. 4 are
shown and further discussed separately in Fig. 5a and b
for the fungal and the bacterial strains, respectively.
Fig. 5a shows the PC scores referring to the two fungal
strains (F1 and F2) and the corresponding culture medium
(F-blank). The F2 data yield a lower score for the PC2, that
is the fluorescence band is broader than the other samples.
On the contrary, the fungal strain F1 does not show any
fluorescence feature since it cannot be distinguished from
the corresponding culture medium (F-blank), as it was also
inferred from the comparison of the fluorescence spectral
shapes (refer to Fig. 2a).
Fig. 5b shows the PC scores referring to the investi-
gated bacterial strains and the corresponding culture med-
3
B1-a
PC1
2.5 B1-b
PC2
B2-a
2 B2-b
B-blank
1.5 F1
F2
1
PC2 score
F-blank
0.5
0
-0.5
-1
-1.5
-2
-6 -4 -2 0 2 4 6 8 10
700 PC1 score
Fig. 4. The two first principal component scores plotted against each
other (PC2 score versus PC1 score). The PCs were obtained applying the
PCA to the complete set of data normalised to the STD.
224 V. Raimondi et al. / Optics Communications 273 (2007) 219–225

0.4 rower fluorescence band in the whole with respect to all

F1 other samples. It is also noteworthy that, although the
0.2 F2
F-blank
data processed with the PCA were normalized, the fluo-
0 rescence spectra of the B1-a sample were the only data
showing a remarkable enhancement of the fluorescence
-0.2 intensity at 450 nm (refer to Fig. 1b).
The same data were also processed with the PCA tech-
PC2 score

-0.4
nique separately for the fungi and the bacteria samples
-0.6 including the corresponding blanks. Results are analogous
to those presented here and obtained by applying the
-0.8 method to the complete data set.
Fluorescence spectra for the fungi and the bacteria sam-
-1
ples were also processed with CA technique. The latter acts
-1.2 as an unsupervised learning classification method where the
number of clusters is not allocated a priori. Bacteria and
-1.4 fungi spectra were normalised and then processed sepa-
-2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2
PC1 score rately with CA according to the procedure described in
Section 2.4. The results are shown as dendrograms for
3
B1-a
2.5 B1-b
B2-a
2 B2-b
B-blank 2.2
1.5
Minimum distance between clusters

2
1
PC2 score

0.5 1.8

0 1.6

-0.5 1.4
-1
1.2
-1.5
1
-2
-6 -4 -2 0 2 4 6 8 10
0.8
PC1 score

Fig. 5. The PC1 and PC2 scores plotted separately for: (a) the fungi and 0.6
F1 F-blank F1 F1 F-blank F2 F2 F2
(b) the bacteria samples.

4.5
ium (B-blank). The elaboration results point out the use-
4
fulness of the method to differentiate among the strains
Minimum distance between clusters

and to separate the contribution of the culture medium: 3.5

data referring to each sample are grouped differently in
the PC1 and PC2 score plot. All the examined strains 3
are well separated from the culture medium as well as
from the other samples. The only exception are the data 2.5
referring to the B2-a and B2-b samples that, although still
2
separated, are almost grouped in the same area. This
leads to think that the two pure cultures can be two 1.5
strains of the same species. The B1-b sample has the high-
est PC1 score that is related to an enhancement in the red 1
region of fluorescence spectrum. The other samples show
the opposite trend with respect to the culture medium, 0.5
that has a significant lower PC1 score. In addition, the B1-a B1-a B2-a B2-a B2-b B2-b B1-b B1-b B1-bB1-bB-blankB-blank
B1-a sample has a high PC2 score, the latter being related Fig. 6. Hierarchical Cluster Analysis applied to: (a) fungi data and (b)
to an enhancement of the fluorescence contribution in the bacteria data. Euclidean distance was used as index of dissimilarity and
central band centred around 450 nm. This yields a nar- single linkage (nearest neighbour) as linkage method.
 V. Raimondi et al. / Optics Communications 273 (2007) 219–225
the fungi and the bacteria samples in Fig. 6a and b, respec-
tively. These results fully confirm the results obtained with
the PCA (refer to Fig. 5): Fig. 6a shows clearly that the
fungi sample F2 can be distinguished from the correspon-
dent blank (F-blank), while the F1 sample cannot be sepa-
rated from its blank. As far as the bacteria samples are
concerned, all strains can be distinguished from the corre-
spondent blank (B-blank). In addition, as pointed out also
by the PCA results, the two strains B2-a and B2-b cannot
be distinguished from each other on the basis of these
results.
4. Conclusions
The results demonstrate the feasibility of the remote
detection in the outdoor of fluorescence features related
to fungal and bacterial strains with a fluorescence LIDAR.
PCA- as well as CA-based data processing can be used to
differentiate among genera and even strains opening good
prospects for the differentiation of heterotrophic organisms
with remote sensing techniques in the field. Although the
detection of heterotrophic biodeteriogens fluorescence
directly on monuments surface can be affected by the fluo-
rescence features of the substrate, the proposed methods
offer a means to overcome this problem since they are
not very sensitive to a fluorescence background of a known
spectral shape, as it was actually that of the culture
medium.
The feasibility of acquiring remotely laser-excited in vivo
autofluorescence of different bacterial and fungal strains in
the outdoor, directly on the monuments, would be particu-
larly valuable since it avoids the collection of samples, their
manipulation and cultivation that, besides being time-con-
suming, can ‘pollute’ the sample itself giving a partially
incorrect results with respect to the actual situation in the
field. Although the experiment was specifically targeted to
the in situ investigation of the biodeterioration process on
monuments in the outdoor, the results can also play a sig-
nificant role in other related environmental fields where an
in situ detection and characterisation of bacterial and fun-
gal strains is required.
In the near future a set of fluorescence measurements
will be carried out directly on stone monuments featuring
areas affected by heterotrophic microorganisms to verify
the potentials of the proposed method.
225
Acknowledgements
This project was supported by the EU-funded Culture
2000 Project (Contract No. CLT 2003/A1/RO-515). The
authors wish to thank Dr. Roxana Radvan and Dr. Rox-
ana Savastru for their support all through the measurement
campaign.
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