Spectrochimica Acta Part B 147 (2018) 132–140

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Spectrochimica Acta Part B

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

The use of micro-energy dispersive X-ray fluorescence spectrometry T

combined with a multivariate approach to determine element variation and
distribution in tobacco seedlings exposed to arsenate
⁎ ⁎
G. Capobiancoa, P. Brunettib, G. Bonifazia, P. Costantinob,c, M. Cardarellib, , S. Serrantia,
a
Dip. Ingegneria Chimica Materiali Ambiente, Sapienza Università di Roma, Italy
b
IBPM-CNR c/o Dip. Biologia e Biotecnologie, Sapienza Università di Roma, Italy
c
Dip Biologia e Biotecnologia, Sapienza Università di Roma

A R T I C LE I N FO A B S T R A C T

Keywords: Here, we present a new scheme of analysis combining micro-energy dispersive X-ray fluorescence spectrometry
X-ray fluorescence spectrometry (μ-XRF) with a multivariate approach that allows to establish the inter-correlation of multiple elements and their
Multivariate approach elemental map in plants. The main advantage of this procedure is that XRF spectral profiles can be analysed
Nicotiana tabacum seedlings directly, by means of principal component analysis (PCA), allowing a quick interpretation of the results.
Arsenic distribution
Furthermore, this analysis requires small amounts of plant material and can be performed in whole individual
Elemental map
seedlings in the hydrated state without chemical extraction. With this technology, we determined the dis-
tribution of arsenic (As) and the variation and spatial distribution of multiple elements in whole tobacco
seedlings grown in the presence of different arsenate concentrations. We observed that As is detectable mainly in
roots, primarily in the basal part, but also in the root apex in seedlings grown in the highest arsenate con-
centration. The low rate of As translocation from roots to shoots and the significant increase in S are consistent
with previous evidence showing that As is retained in roots by forming complexes with thiol peptides. We also
found a significant non-linear increase in P, as arsenate is taken up by phosphate transporters and induces the
expression of genes encoding them. A decrease in Mn, Fe and Zn proportional to the accumulation of As and in
the same tissues, suggests a competition of these elements with As for cellular transporters.

1. Introduction imaging analysis was applied to characterize the simultaneous sub-

Micro X-ray fluorescence (μ-XRF) is a valuable and sensitive tool for
the analysis of the distribution of elements in different regions of a
plant. In recent years this technique has been used in combination with
or has often replaced conventional approaches. The quantitative de-
termination of multiple elements in plants has been generally per-
formed by acid digestion of the samples followed by ICP-OES analysis
(inductively coupled plasma-optical emission spectrometer) [1,2]. This
approach requires large amounts of plant material and is therefore
performed on a limited number of samples from removed plants and it
does not give any information about the topological distribution of
elements. Analyses of plant tissues by synchrotron X-ray fluorescence
techniques indeed drastically reduced the amount of required material
but mainly dehydrated tissues are utilized [3]. Since X-rays can be fo-
cused to spots smaller than 1 mm, researchers have used it to identify
and quantify nutrients and non-essential elements such as arsenic (As)
or cadmium (Cd) [4]. Synchrotron-based X-ray microfluorescence
cellular distribution of a number of mineral elements. This fine-imaging
method can reveal whether these elements colocalize [5–6]. Very re-
cently a μ-XRF instrument has been designed and applied for in vivo
analysis of Arabidopsis leaves- not detached from the plant- aiming to
reduce as much as possible damages to the plant tissues [7].
μ-XRF technique has proved particularly effective in the study of
elemental distribution in different organs of plants exposed to different
elemental contaminants [8,9,10].
The most frequent contaminants of soils are heavy metals, including
As, and among them As is recognized as one of the main toxicants
worldwide. Arsenic is released into the environment both by natural
processes, such as weathering of rocks, and by anthropogenic activities
such as mining or the use of As-containing pesticides, herbicides and
wood preservatives [11]. The accumulation of As in the biosphere and
in the food chain has serious effects on the environment and human
health [12].
Plants have widely different capabilities of tolerating and

⁎
Corresponding authors.
E-mail addresses: maura.cardarelli@uniroma1.it (M. Cardarelli), silvia.serranti@uniroma1.it (S. Serranti).

https://doi.org/10.1016/j.sab.2018.05.029
Received 21 September 2017; Received in revised form 28 May 2018; Accepted 30 May 2018
Available online 30 May 2018
0584-8547/ © 2018 Elsevier B.V. All rights reserved.
G. Capobianco et al.
Fig. 1. Nicotiana tabacum: control samples (A), 50 μM As (B) and 100 μM As (C). (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
accumulating As, depending on the amount of this element that is taken
up from the soil and on how the plant distributes it in its tissues [13].
Some hyper-accumulator plants such as the fern Pteris vittata are cap-
able of accumulating As in their aerial part, thus tolerating high con-
centrations of this soil pollutant [14]. Other plants, including crops,
accumulate As mainly in roots and show limited toxicity symptoms
[15].
So far μ-XRF has been focused mostly on hyperaccumulators, to
provide both in a semi-quantitative and topological way a better de-
scription of the accumulation mechanisms of As. A synchrotron X-Ray
study on Pteris vittata, describes the accumulation and spatial dis-
tribution of As in different organs, detached from the fern [16]. In
addition, benchtop μ-XRF has been successfully used on the hyper-
accumulator Pityrogramma calomelanos to determine the distribution of
As in the hydrated state as well as As and P content on pelletized
powdered samples from the pinna, stipe and root. This study showed
that As causes alterations in P distribution [17].
However As distribution, as well as changes in multiple elements,
determined in hyperaccumulator organs, is not sufficient alone to per-
form a full understanding of metabolic processes that occur within
plants, including crops, exposed to As, while this can dramatically
contribute to develop techniques limiting As migration into the food
chain. The mechanisms operating in plants for As uptake, transport and
cellular distribution can be studied by measuring the changes in the
localization and concentration of multiple elements [18]. It is important
to simultaneously assess the variations in the concentration and loca-
lization of essential elements in different tissues of an entire plant ex-
posed to As.
In this study we focused on Nicotiana tabacum whole individual
seedlings exposed to arsenate, whose As content has been previously
quantified by ICP-OES and the concomitant production of
Phytochelatin-S-rich peptides -responsible for As detoxification- has
been demonstrated [19]. The localization of As and multiple elements
as well as a semi-quantitative evaluation or their content was in-
vestigated by μ-XRF combined with a multivariate approach, allowing a
direct and fast analysis of elemental spectral profiles. The experimental
data to be submitted to multivariate analysis usually derive from the
computation of quantitative parameters (e.g., peak areas or peak
heights) for each identified chemical element within the spectrum. But
with this method the processing time is considerable. Such an approach
is time consuming and important information, characterising the
spectral profiles, may be lost [20]. The proposed approach was aimed at
developing an appropriate strategy for multivariate treatment of XRF
spectral and hypermaps data starting from the analysis of the raw data.
The improvement of this strategy, able to account for inter-correlation
among elements detected, may represent an important step forward in
plants analyses. To reach this goal Principal Component Analysis (PCA)
is applied. PCA is a multivariate analysis method identifying the lowest
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Spectrochimica Acta Part B 147 (2018) 132–140
number of independent uncorrelated variables Principal Components
needed to explain the maximum variance of a large set of data [21].
With reference to tobacco seedlings exposed to arsenate, the results are
visualized as eigenimages or eigenvectors representing the distribution
variance of detected elements in the plants, as well as As concentration
with respect to the detected elements.
2. Experimental
2.1. Seedling growth
Nicotiana tabacum Petite Havana SR1 seeds were surface sterilized
by immersion in sodium hypochlorite solution containing 1% available
chlorine (v/v), for 5 min. Seeds were then washed four times with
sterile distilled water, stratified at 4 °C for three days and then in-
cubated in half-strength MS medium (Duchefa M0222), supplemented
with 8% agar (Duchefa P1001) and 2% sucrose (Duchefa 50,809), in a
growth chamber in a 16/8 h light/dark cycle at 22 °C. Seven days after
germination 10 seedlings were transferred to the medium containing 0,
50, 100, μM Na2HAsO4·7H20 (Sigma A6756). After 9 days seedlings
were collected for subsequent analysis (Fig. 1).
2.2. μ-XRF analysis and statistical approach
A μ-XRF benchtop spectrometer (M4 Tornado, Bruker®), equipped
with a Rh X-ray tube with polycapillary optics and XFlash® detector
providing an energy resolution of better than 145 eV, was utilized to
perform the analysis. The polycapillary optics allow the focusing of tube
radiation, on a very small spot size (about 30 μm), by multiple reflec-
tions thus providing a spot intensity increase by up to 104x compare to
collimator, the results being faster and more detailed (i.e. spatial re-
solution) analysis. Spectrum energy calibration was performed daily,
before each analysis batch, by using zirconium (Zr) metal (Bruker®
calibration standard). The sample chamber can be evacuated to
25 mbar and, therefore, light elements such as sodium can be measured.
The samples are mounted on a thin layer (0.01 mm) of polyethylene,
suspend on chamber to reduce the noise of map acquisition and pressed
parallel to the stage table. Samples with an even and flat surface like
sections can be put directly on the stage table without coating. At
constant exciting energies of 50 kV and 500 μA, the measurement
conditions were adapted for the different investigated samples. The
dwell time and the pixel resolution, depending on the samples size [20]
have been respectively set up at 15 ms/pixel and 35 μm. An image
showing the relative element map was realised (Fig. 2).
The hypermap import and processing have been preliminary per-
formed utilizing the ESPRIT M4 Tornado software and, after the ex-
traction of raw data, statistical analysis was performed following a
standard chemometric based approach utilizing the PLS_Toolbox
G. Capobianco et al.
Fig. 2. Nicotiana tabacum hypermap obtanied by M4 tornado: control samples (A), 50 μM As (B) and 100 μM As (C). (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)
CONTROL
As TREATED 50
As TREATED 100
Fig. 3. Mosaic image of μ-XRF hypermap of As-treated Nicotiana tabacum
seedlings after background removal. As-treated 50 (50 μM As) and As treated
100 (100 μM As). (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
(Version 7.8 Eigenvector Research, Inc.) running inside Matlab (Version
7.11.1), The Mathworks, Inc. Principal Component Analysis (PCA) was
then carried out to identify correlations among sample features, de-
tected by classical analyses, and corresponding to hypermap features
[21,22]. In order to assess the variations between control and As treated
samples, the spectral attribute collected for each pixel constituting the
investigated samples were combined into a single hypermap (i.e. mo-
saic image) (Fig. 3) [23]. Data were then pre-processed to highlight
sample spectral differences and to reduce the impact of possible ex-
ternal sources of variability, allowing a more accurate interpretation of
the results obtained by the classification modelling. Different pre-pro-
cessing algorithms were applied such as Baseline, Normalize,
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Spectrochimica Acta Part B 147 (2018) 132–140
Smoothing and Mean Centering (MC) in order to reduce unwanted ef-
fects due to the background, reducing noise and transforming the data
in such a way to stress and highlight linear relationship between signals
and concentrations [24–26]. In the following synthetic description of
the pre-processing algorithms, in respect of their utilization in this
work, is reported.
2.2.1. Baseline
This procedure was applied to perform a fitting of a polynomial of a
specific order to points, which are known to be baseline (no-signal)
points. This approach is commonly utilized in spectroscopic applica-
tions where the signal in some variables is due only to baseline
(background) [27].
2.2.2. Normalize
Following this approach, the value of each variable was divided by
the absolute value of all the variables [28].
2.2.3. Smoothing
The most common technique of differentiation for smoothing is the
Savitzky–Golay routine [29]. This routine can be used for smoothing/
noise reduction, in order to avoid amplification of high-frequency noise
during the derivation process as it happens in the case of finite differ-
ence derivation [30].
2.2.4. Mean Centering (MC)
Centering and scaling are the two most common types of pre-pro-
cessing, normally applied before PCA. MC has the effect of including an
adjustable intercept in multivariate models and is suitable for con-
tinuous data, such as hyperspectral data. For example, Mean Centering
both X and Y blocks in a regression model effectively allows for a
nonzero intercept of the regression line [31].
3. Results and discussion
3.1. Determination of As and essential elements in roots and leaves of
tobacco seedlings
To analyse the level of As and essential elements in different organs
of tobacco seedlings exposed for 9 days to 0, 50, 100 μM arsenate, ten
different, Specific Areas in the Sample (SAS) - utilized to collect the
spectra in the roots and leaves of each seedling - were selected. Fig. 4
shows an example of μ-XRF spectrum obtained from the analysis of
Nicotiana tabacum seedlings relative to plants exposed to 100 μM As.
Spectra obtained from each SAS contain the characteristic Kα of As
(10.54 keV), as well as of Zn, Fe, Mn, Ca, K, S, P, Mg and Si. Semi-
quantitative results obtained by deconvolution are summarized in
G. Capobianco et al. Spectrochimica Acta Part B 147 (2018) 132–140

Fig. 4. Fragment of a typical Energy Dispersive micro X-Ray Fluorescence (μ-EDXRF) spectrum related to Nicotiana tabacum seedlings exposed to 100 μM As.

Table 1. Arsenic was not detectable in seedlings grown without ar-
senate, while in seedlings grown at 50 μM As it was detectable only in
the roots. When grown at 100 μM As, seedlings showed a significantly
higher level of As in roots and a lower level in leaves. In these seedlings,
the Translocation Factor (TF, the ratio of As in shoots vs roots) was
0.14.
Almost all the above mentioned essential elements showed sig-
nificant variations from controls in As-treated seedlings; in particular, a
nearly linear decrease of Fe, Mn, Zn was observed in roots at increasing
arsenate; in contrast a significant, non-linear increase in P, and S was
observed in roots at increasing arsenate (Table 1).
These results point to an impact of As exposure on the levels of most
essential elements and suggest a main effect on Fe, Mn, Zn, P and S.
3.2. Identification of As and essential element distribution in tobacco
seedlings
To investigate the distribution of elements in seedlings grown in the
presence or absence of As, micro-chemical qualitative maps - false
colour images where the presence of each specific detected element in
the investigated areas of the samples is topologically assessed - were
obtained. As expected, As was not detectable in seedlings grown in the
absence of arsenate while it was detectable only in roots in As-grown
seedlings. At 50 μM arsenate, As was localized along the root but not in
the apex, and at 100 μM As was also detectable in the apical part of the
roots (Fig. 5). Mn, Fe and Zn were detectable only in roots – in the basal
and in the apical part – both in the presence and in the absence of
arsenate, S, and P although detectable in the absence of As, showed a
stronger signal in roots and leaves of seedlings grown in the presence of
arsenate (Fig. 5). Thus the spatial distribution of As appears to be si-
milar to those of Fe, Zn and Mn, whose levels simultaneously decrease
in the presence of arsenate.
3.3. Multivariate analysis of μ-XRF hypermap
To better understand the variation of different essential elements in
response to As, a mosaic image was built as previously outlined, starting
from the collected spectra datasets after background removal, by means
of a preliminary PCA with Baseline, Smoothing, Normalize, Mean
Centering pre-processing.
PCA score plots of hypermap data show significant changes in dif-
ferent element between controls and As-treated samples (Fig. 6A). In
particular, the main variations were detected in the first and the second
principal components. By analysing the loading plots related to the first
component, we found that variations are mainly due to P and S, while
those in the second component are due to Fe, Mn and Zn (Fig. 6B–C).
To identify the distribution of As in each seedling, the range be-
tween 10.1 and 11.2 keV, corresponding to As Kα, was selected.
Normalize, Smoothing and Mean Centering, for PCA pre-processing was
carried out. As shown in Fig. 7A, the PCA score plots showed three

Table 1
Semi quantitative determination carried out by Energy Dispersive μ-XRF (ED μ-XRF) on root and leaves of Nicotiana tabacum for control (As0) and As-treated sample.
Mg Al Si P S K Ca Mn Fe Zn As

Roots Weigth (%)

As0 0.23 0.29 0.02 2.12 1.25 78.08 12.09 2.35 2.77 0.79 0.00
Sigma mean: 0.06 0.01 0.01 0.16 0.06 1.23 1.14 0.11 0.19 0.07 0.00
As50 1.55 0.16 0.50 6.98 12.96 68.32 7.76 0.66 0.61 0.43 0.08
Sigma mean: 0.26 0.04 0.11 0.30 0.26 0.64 0.34 0.06 0.07 0.04 0.01
As100 0.58 0.28 0.17 3.98 4.26 70.28 19.24 0.44 0.37 0.25 0.14
Sigma mean: 0.07 0.04 0.10 0.26 0.15 1.44 1.49 0.06 0.04 0.04 0.02

Leaves Weigth (%)

As0 0.31 0.35 0.12 1.01 0.84 85.27 10.94 0.41 0.42 0.33 0.00
Sigma mean: 0.07 0.05 0.04 0.08 0.07 1.07 0.94 0.04 0.05 0.02 0.00
As50 5.86 0.18 0.16 9.36 7.86 63.48 12.69 0.29 0.08 0.03 0.00
Sigma mean: 0.14 0.03 0.03 0.19 0.09 0.65 0.37 0.01 0.00 0.00 0.00
As100 1.89 0.28 1.04 3.23 6.23 68.41 18.20 0.55 0.11 0.03 0.02
Sigma mean: 0.16 0.03 0.71 0.17 0.33 1.57 1.40 0.05 0.01 0.00 0.00

Different As concentrations: As0: 0 μM arsenate. As50: 50 μM arsenate and As100: 100 μM arsenate.
Sigma means: standard deviation calculated on 10 measurements performed for each root and leaves.

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Fig. 5. μ-EDXRF element maps of As, P, S, Fe, Zn and Mn detected in Nicotiana tabacum controls and As-treated seedlings (0, 50, and 100 μM As). (For interpretation
of the references to color in this figure legend, the reader is referred to the web version of this article.)

different clouds corresponding to 0, 50 and 100 μM arsenate, respec-
tively. The analyses of all seedlings, carried out at the same time, re-
vealed As mainly in the basal region of the roots at 50 μM arsenate
while the signal was stronger and detectable also in root apex at 100 μM
arsenate (Fig. 7B).
In order to analyse Zn, Fe and Mn accumulation in root regions with
a high As accumulation, we selected pixel/spectra where As signal is
the highest, i.e. the basal part of the root (Fig. 8), and we analysed
spectral ranges from 6 to 12 keV to compare the As peak with those of
Fe, Mn and Zn.

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Fig. 6. Principal Component Analysis (PCA) score plot of Nicotiana tabacum hypermap (A), loadings of PC1 and PC2 (B) and PCA score plot with the set classes (C).
(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 7. PCA score plot related to the seedlings by different As concentrations
(As0: 0 μM As, As50: 50 μM As and As100: 100 μM As) (A). Image of μ-EDXRF
hypermap with class selected: controls and As-treated seedlings (0, 50, and
100 μM As) (B). (For interpretation of the references to color in this figure le-
gend, the reader is referred to the web version of this article.)
Spectra analysis was performed before (Fig. 9A) and after (Fig. 9B)
pre-processing with Baseline and Smoothing. Raw data showed a sig-
nificant variance of As peak vs Fe, Mn and Zn peaks, while pre-pro-
cessing analysis showed a linear relationship between As increase and
Fe, Mn and Zn decrease. The corresponding PC1-PC2 score and loading
plots (Fig. 10A, B) further support the previous considerations. To
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Spectrochimica Acta Part B 147 (2018) 132–140
analyse P and S nonlinear increase in root regions with a high As, we
analysed by PCA the spectral ranges from 1.8 to 2.4 keV (P and S Kα)
and from 10 to 10.7 keV (As Kα). Fig. 10C, D, E and F report the PCA
score and loadings plots of AseP and AseS, respectively. In both AseP
and AS-S score plots the dispersion of the points generating each class
cloud are clearly detectable and increase with increasing As con-
centrations.
The AseP loadings plot (Fig. 10D) shows that the dispersion of
sample As 50 μM is mainly influenced by P variance (PC2 positive va-
lues). On the contrary, sample As 100 μM is influenced by As increase
(PC1 positive and PC2 negative values).
The AseS loadings plot (Fig. 10F) shows that the dispersion of
sample As 50 μM is caused by S variance (PC1 positive values and PC2
negative values). Sample As 100 μM is mainly influenced by As increase
(PC2 positive values).
3.4. Biological significance of the distribution of As and essential elements
Comparative analysis of As accumulation in roots and leaves of to-
bacco seedlings confirms that this species is not a hyperaccumulator
because as is taken up by, and accumulated predominantly in root cells.
Accordingly, its TF is 0.14 comparable to that of other non-hyper-
accumulators such as Brassica juncea [32] and rice [33].
This TF value is in good agreement with previously published re-
sults obtained by using ICP-OES to measure As content in roots and
shoots of N. tabacum seedlings grown in the presence of As 50, 100 and
200 μM [19]. In addition, our elemental map shows that As accumulates
G. Capobianco et al. Spectrochimica Acta Part B 147 (2018) 132–140

As
As

As

Fig. 8. Specific Areas in the Sample (SAS) selected on the Nicotiana tabacum hypermaps characterized by different As concentrations. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 9. Mean spectra of SAS selected on Nicotiana tabacum, grown in the presence of different As, before (A) and after pre-processing (i.e. Baseline and Smoothing)
(B). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

mainly in the basal region mostly on of the roots and it is detectable in
the root apex only at high arsenate concentrations, indicating that As is
transported basipetally.
We found a significant increase of P in roots of low As-grown
seedlings and, to a lesser extent, in roots of high As-grown seedlings. It
is known that in many species such as Arabidopsis and rice As (V) and P
compete for uptake by the same transporters [34]. Thus, the increase in
P suggests that As (V) (the As present in our growth medium) uses P
transporters also in tobacco seedlings where it possibly induces their
synthesis like in Pteris vittata [35]. This increase in P transporters- that
exhibit a preference for P over AsV (Meharg & Macnair, 1990; Meharg,
1994)- can lead to an increase in P uptake. This increase in P accu-
mulation is lower at high As, possibly due to the increasing competition
between As and P as As concentrations become higher.
The increase in S is probably caused by the synthesis of glutathione
and phytochelatins, which is known to occur particularly in roots of
tobacco seedlings in response to Cd and As. PCs, represent the main
detoxification system in plants, which acts by forming PC-As complexes
with As (III) (arsenite) that are compartmentalized into the vacuole. In
our hands, the increase in S is higher at low than at high As con-
centrations, consistent with previous results showing that PC synthesis
increases up to a threshold level of As then decreases at higher As
concentrations [36]. Our results corroborate the hypothesis that non-
hyperaccumulator plants do not translocate efficiently As to the shoots,
because it is stored in root cells as PC-As complexes [37].
Elemental analysis and map combined with a multivariate approach
allowed us to show a linear and concomitant decrease in Fe, Mn and Zn
in As-grown seedlings. In particular, their levels are severely decreased
in the basal region of the roots where As preferentially accumulates,
This depletion suggests that inside the cells As compete with Fe, Mn and
Zn for binding to the metal tolerance proteins (MTP) which are low-
specificity efflux transporters that regulate homeostasis of these metals

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Fig. 10. PCA score plots (A) and loadings (B) of SAS with high As concentration of Nicotiana tabacum. PCA score plots (C) and loadings (D) of P and As concentration
in Nicotiana tabacum. PCA score plots (E) and loadings (F) of S and As concentration in Nicotiana tabacum. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)

[38]. the localization of As and the investigated elements in the whole

4. Conclusions
This study proposes an analytical method based on micro-energy
dispersive X-ray fluorescence spectrometry (μ-XRF) for semi-quantita-
tive determination and mapping of As and essential elements, as uti-
lized in tobacco seedlings. This technique allows the utilization of a
small quantity of hydrated plant material such as individual 16-days-
old tobacco seedlings in a non-destructive method, since it does not
require chemical extraction. The combined multivariate approach to-
gether with the acquisition set up allowed us to obtain a ‘snapshot’ of
seedlings at the moment of data acquisition. By means of this tech-
nology -in particular through PCA- we could easily detect differences
between seedlings grown with As and those without, and perform an
objective analysis of the data especially for low-concentration elements.
In particular, it was possible to show the variation of some elements
between plants grown in the presence or absence of As, and to verify the
maximum accumulation of As in the roots. We established significant
correlations between As exposure and the increase of light elements
such as P and S that are constituent of transport proteins and detox-
ification peptides, respectively. In addition, the selection of peaks re-
lated to Fe, Zn and As permits to confirm the correlation between As

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increase and the decrease of Fe and Zn in Nicotiana tabacum seedlings.
The element map obtained allows to identify the distribution of As
in roots where it is localized only in the basal part al low As con-
centrations, and confirms its absence in leaves. Our approach can be a
powerful tool to investigate the effects of As and (possibly) other metal
pollutants and to compare the response to these toxicants between
different plant species.
The results obtained are also very promising in the perspective of
large and systematic environmental surveys, since the proposed ap-
proach allows to analyse a higher number of samples in less time than
with the techniques currently adopted, with a good identification of
contaminated and uncontaminated plants.
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