Journal of
Imaging
Article
Computer Assisted Examination of Infrared and Near
Infrared Spectra to Assess Structural and Molecular
Changes in Biological Samples Exposed to Pollutants:
A Case of Study
Mauro Mecozzi 1, * and Elena Sturchio 2
1
2
*
Laboratory of Chemometrics and Environmental Applications, ISPRA, Via di Castel Romano 100,
00128 Rome, Italy
Italian Workers’ Compensation Authority (INAIL), Department of Technological Innovation,
Safety Plants and Products and Anthropic Settlements (DIT), Via Roberto Ferruzzi 38/40, 00143 Rome,
Italy; [email protected]
Correspondence: [email protected] or [email protected]; Tel.: +39-6-5007-3287
Academic Editors: Carosena Meola and Gonzalo Pajares Martinsanz
Received: 14 October 2016; Accepted: 8 February 2017; Published: 16 February 2017
Abstract: We present a computer assisted method for the examination of the structural changes
present in the probe organism Vicia faba exposed to inorganic arsenic, detected by means of Fourier
transform infrared (FTIR) and Fourier transform near infrared (FTNIR) spectroscopy. Like the
common ecotoxicological tests, the method is based on the comparison among control and exposed
sample spectra of the organisms to detect structural changes caused by pollutants. Using FTIR
spectroscopy, we measured and plotted the spectral changes related to the unsaturated to saturated
lipid ratio changes (USL), the lipid to protein ratio changes (LPR), fatty and ester fatty acid content
changes (FA), protein oxidation (PO) and denaturation, and DNA and RNA changes (DNA-RNA).
Using FTNIR spectroscopy, we measured two spectral ranges that belonged to hydrogen bond
interactions and aliphatic lipid chains called IntHCONH and Met1overt, respectively. The FTIR
results showed that As modified the DNA-RNA ratio and also caused partial protein denaturation
in the Vicia faba samples. The FTNIR results supported the FTIR results. The main advantage of the
proposed computational method is that it does not require a skilled infrared or near infrared operator,
lending support to conventional studies performed by toxicological testing.
Keywords: FTIR spectroscopy; FTNIR spectroscopy; computer analysis; toxic effect of As; structural
changes in organisms
1. Introduction
Modern ecotoxicology is focused on the study of the interactions among organisms and pollutants
with special emphasis on the comprehension of their biological response to pollutant exposures.
In these fields of study, novel techniques based on vibrational spectroscopy such as Fourier transform
infrared (FTIR) and Fourier near infrared (FTNIR) have been proposed because the biomolecules of
the living organisms consist of polysaccharides, proteins, and lipids, molecules that have significant
FTIR and FTNIR absorptions [1]. Table 1 reports the list of the most significant FTIR bands of the
biomolecules present in Vicia faba roots.
When the living cells are exposed to pollutants, the toxic effect causes structural changes in the
biomolecules depending on their interaction with the pollutants [1–6]. Using vibrational spectroscopic
techniques such as FTIR and FTNIR, these changes can be visualized by means of band shifts, band
shape, and peak intensity (both increase or decrease) changes. Then, the visual comparison among
J. Imaging 2017, 3, 11; doi:10.3390/jimaging3010011
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J. Imaging 2017, 3, 11
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band shifts, band shape, and peak intensity (both increase or decrease) changes. Then, the visual
2 of 13
the control and exposed samples (Figures 1 and 2), such as a common
toxicological and cytotoxic test, becomes a tool for identifying the typical structural changes in the
exposed organism.
the control and exposed samples (Figures 1 and 2), such as a common toxicological and cytotoxic test,
becomes a tool for identifying the typical structural changes in the exposed organism.
J. Imaging
2017, 3, 11
comparison
among
Table 1. List of the most significant infrared (FTIR) bands and related functional groups assigned
for Vicia faba samples. The “*" bands are evidenced by means of second derivative spectroscopy.
Table 1. List of the most significant infrared (FTIR) bands and related functional groups assigned for
−1
Vicia faba
samples. The “*"
are evidenced by means
of secondGroup
derivative spectroscopy.
Wavenumber
cmbands
Functional
3350–3450
−1
Wavenumber
3200–3250cm
3350–3450
3010–3020
3200–3250
3020–3060
3010–3020
2850–2950
3020–3060
2100–2500
2850–2950
2100–2500
1730–1740
1730–1740
1700–1715
1700–1715
1620–1670
1620–1670
1670 *
1670 *
1650 *
1650 *
1635
1635 **
1625–1630
*
1625–1630 *
1540–1550
1540–1550
1510
1510 **
1400–1460
1400–1460
1350–1440
1350–1440
1240–1340
1240–1340
1120–1160
1120–1160
1085–1080
1085–1080
1080–1060
1080–1060
900–800
900–800
OH of carbohydrates, proteins, and polyphenols
Group
NH2 Functional
aminoacidic
group
OH of carbohydrates,
proteins,
and polyphenols
CH alkene
group
NH
aminoacidic
group
2
CH of aromatic ring
CH alkene group
CH and CHCH
2 aliphatic stretching group
of aromatic ring
C=CCH
conjugated
and C≡C
CH and
2 aliphatic stretching group
C=C
conjugated
andgroup
C≡C
C=O
ester
fatty acid
C=O
group
C=Oester
fattyfatty
acidacid
group
C=O fatty acid group
C=O Amide I band
C=O Amide I band
beta turns Amide I band
beta turns Amide I band
alpha helix Amide I band
alpha helix Amide I band
beta
sheet
beta
sheetAmide
Amide II band
band
random
coil
Amide
I
band
(i.e.,
denaturation
of proteins)
random coil Amide I band (i.e., denaturation of proteins)
C–N
C–NAmide
AmideIIII band
band
Lignin
skeletal
band
Lignin skeletal band (aromatic)
(aromatic)
stretching –C=O inorganic carbonate
stretching –C=O inorganic carbonate
CHCH
and
CH
2 aliphatic bending group
and
CH
2 aliphatic bending group
C–N
C–NAmide
AmideIII
III band
band
C–O–C
polysaccharide
and
DNA
and RNA
C–O–C polysaccharide and DNA and
RNAbackbones
backbones
P=O
phospholipids
in
DNA
and
P=O phospholipids in DNA and RNA
RNA
C–O carbohydrates in DNA and RNA backbones
C–O
carbohydrates
in DNA and
backbones
C=C,
C=N,
C–H in ring structure,
DNARNA
and RNA
backbones
C=C, C=N, C–H in ring structure, DNA and RNA backbones
FTNIR
FTNIRspectroscopy
spectroscopygenerally
generallyhas
hasa alower
lowerinformation
informationpower
powerthan
thanFTIR
FTIRspectroscopy,
spectroscopy,because
because
the
absorptions
of
the
functional
groups
are
less
intense
(two
orders
of
magnitude
at
least)
than
the absorptions of the functional groups are less intense (two orders of magnitude at least) thanthe
the
corresponding
absorptions
ofof
the
same
functional
groups
inin
the
infrared
region
[7].[7].
corresponding
absorptions
the
same
functional
groups
the
infrared
region
Figure
Exampleofof
control
(black),
exposed(purple),
(purple),and
anddifference
difference(green)
(green)FTIR
FTIRspectra
spectraofof
Vicia
Figure
1. 1.
Example
control
(black),
exposed
Vicia
-1
faba
samples
treated
with
As.The
Thenegative
negativevalues
valuesobserved
observedfor
forthe
thedifference
differencespectrum
spectrum
faba
samples
treated
with
3030
g Lg−L1 As.
inin
−1 show a reduction of the molecular absorptions in the exposed
−
1
the
range
between
3100
and
1400
cm
the range between 3100 and 1400 cm show a reduction of the molecular absorptions in the exposed
samples
with
respect
the
control
sample.
The
two
black
arrows
show
the
range
where
a flat
water
samples
with
respect
toto
the
control
sample.
The
two
black
arrows
show
the
range
where
a flat
water
band
could
be
observed
in
the
case
of
a
non-efficient
lyophilisation.
band could be observed in the case of a non-efficient lyophilisation.
J. Imaging 2017, 3, 11
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However,FTNIR
FTNIRspectroscopy
spectroscopyhas
has some
some advantages
advantages with
with respect
respect to
to FTIR
FTIR spectroscopy.
spectroscopy. In
In fact,
fact,
However,
the
absorptions
of
–OH
(carbohydrate,
protein,
and
lipid)
and
–NH
(protein)
groups
are
better
the absorptions of –OH (carbohydrate, protein, and lipid) and –NH (protein) groups are better resolved
resolved
those
inregion
the FTIR
and the bond
hydrogen
bond interactions
shifts
than
thosethan
in the
FTIR
andregion
the hydrogen
interactions
cause largercause
bandlarger
shifts band
than those
than
those
observed
in
the
FTIR
region,
making
their
interpretation
easier
[7].
This
makes
FTNIR
observed in the FTIR region, making their interpretation easier [7]. This makes FTNIR spectroscopy
spectroscopy
reliable for ecotoxicological
studies itasprovides
well, because
it provides
complementary
reliable
for ecotoxicological
studies as well, because
complementary
information
to that
information
to
that
obtained
by
FTIR
spectroscopy
[8,9].
obtained by FTIR spectroscopy [8,9].
Figure2.2.Example
Exampleofof
control
(black),
exposed
and difference
near infrared
FTNIR
Figure
control
(black),
exposed
(red),(red),
and difference
(green)(green)
near infrared
FTNIR spectra
−1 negative
for
a Vicia
sample
20 g·with
L−1 As.
The
values observed
in the range
between
As. The negative
values observed
in the
range
spectra
forfaba
a Vicia
fabatreated
samplewith
treated
20 g·L
−1 of
7500
and 4000
the difference
spectrum
showspectrum
a reduction
of the
in the
difference
show
a molecular
reduction absorptions
of the molecular
between
7500cm
and
4000
cm−1 of the
exposed
samples
respect
to the control
sample.
absorptions
in thewith
exposed
samples
with respect
to the control sample.
Figure22reports
reportsan
anexample
exampleofofthe
the
comparison
between
FTNIR
spectra
of the
control
Figure
comparison
between
thethe
FTNIR
spectra
of the
control
andand
As
As
exposed
Vicia
faba
samples,
while
Table
2
reports
the
detailed
list
of
the
near
infrared
bands.
exposed Vicia faba samples, while Table 2 reports the detailed list of the near infrared bands.
However, the
the FTIR
FTIR and
1 and
2 are
characterized
by the
However,
and FTNIR
FTNIR spectra
spectrasuch
suchasasthose
thoseininFigures
Figures
1 and
2 are
characterized
by
presence
of
several
bands
and
the
interpretation
of
the
structural
changes
caused
by
a
pollutant
the presence of several bands and the interpretation of the structural changes caused by a pollutant
exposure isis not
not an
an easy
easy task.
task. In
In fact,
fact, aa recent
recent review
review describing
describing the
the use
use of
of FTIR
FTIR spectroscopy
spectroscopy to
to
exposure
investigatephysiological
physiologicalcell
cellstates
statesunderlines
underlines
complex
aspects
of their
interpretation,
because
investigate
thethe
complex
aspects
of their
interpretation,
because
the
the
extraction
of
information
about
content
and
structure
in
biological
systems
requires
skilled
extraction of information about content and structure in biological systems requires skilled operators
operators
and sometimes
specific
tools
such as chemometric
methods [10].
and
sometimes
specific tools
such as
chemometric
methods [10].
Table2.2.List
Listofof
most
significant
FTNIR
bands
related
functional
groups
assigned
for
the
Table
thethe
most
significant
FTNIR
bands
andand
related
functional
groups
assigned
for the
Vicia
Vicia
faba
samples.
faba samples.
Wavelength (cm−1)
Functional Group
Functional Group
8400–8800
–CH and –CH2 second overtone aliphatic chains
8400–8800
–CH
and
–CH
overtone aliphatic
chains
2 second proteins
6600–6900
–OH
first
overtone
and lipids
6600–6900
–OH first overtone proteins and lipids
6500–6600
–NH2 amino acid first overtone
6500–6600
–NH2 amino acid first overtone
5100–5200
Combination
Amide
5100–5200
Combination
AmideI Iand
and–OH
–OH proteins
proteins
4800–5000
Combination
–OH
proteins
4800–5000
Combination
–OHand
andAmide
Amide II proteins
4200–4800
Combination
–NH
,
–CH,
C–C,
and
2
4200–4800
Combination –NH2, –CH, C–C, and –OH
–OH
Wavelength (cm−1 )
With the
the aim
aim of
of making
making the
the interpretation
interpretation of
of FTIR
FTIR and
and FTNIR
FTNIR spectra
spectra of
of biological
biological samples
samples
With
submittedfor
fortoxicological
toxicological
testing
easier,
present
a digital
approach
does
not require
submitted
testing
easier,
we we
present
a digital
approach
whichwhich
does not
require
skilled
skilled
knowledge
for
the
interpretation
of
FTIR
and
FTNIR
spectra.
This
method
is
presented
and
knowledge for the interpretation of FTIR and FTNIR spectra. This method is presented and discussed
discussed
for
the
exposure
of
Vicia
faba
roots
to
inorganic
As.
for the exposure of Vicia faba roots to inorganic As.
Imaging2017,
2017,3,3,11
11
J.J.Imaging
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13
44of
2. Materials and Methods
2. Materials and Methods
2.1.
2.1.Sample
SampleCollection
Collectionand
andPreparation
Preparationfor
forFTIR
FTIRand
andFTNIR
FTNIRSpectroscopic
Spectroscopic Analysis
Analysis
Vicia
Viciafaba
fabawas
wasgrown
grownin
indifferentially
differentiallypolluted
pollutedsoils.
soils.Vicia
Viciafaba
fabaseeds
seedswere
weresowed
sowedin
in250
250ggquartz
quartz
sand
soil
in
aluminum
basins.
Each
basin,
containing
25
seeds,
was
treated
with
40
mL
sand soil in aluminum basins. Each basin, containing 25 seeds, was treated with 40 mL sodium sodium
arsenate
arsenate
dibasic heptahydrate
and incubated
in chamber
a climatic(atchamber
(at60%
21 relative
°C and humidity)
60% relative
dibasic heptahydrate
and incubated
in a climatic
21 ◦ C and
for
humidity)
for
5
days
to
enable
germination.
Seeds
were
grown
in
a
quartz
sand
soil
basin
irrigated
5 days to enable germination. Seeds were grown in a quartz sand soil basin irrigated with 40 mL of
with
mL of water.
Irrigation
with
water
only was
used All
as of
a the
negative
control.
Allrepeated
of the
water.40Irrigation
with water
only was
used
as a negative
control.
experiments
were
experiments
were
repeated
three
times
in
June
2013
and
three
times
in
July
2013.
Concentrations
of
three times in June 2013 and three times in July 2013. Concentrations of As in the irrigation water were
−1
As
in theatirrigation
selected
at 10 and
20 atg·L
for 30
theg·June
exposure
at 20 and
selected
10 and 20water
g·L−1were
for the
June exposure
and
20 and
L−1 for
the Julyand
exposure;
the
−1 for the July exposure; the range was selected because at lower concentration, no one
30
g·L
range was selected because at lower concentration, no one molecular effect was detected whereas at
molecular
effect was detected
whereas
at higher
there was
the inhibition
of growth.
higher concentrations
there was
the inhibition
of concentrations
growth. More details
concerning
the preparation
of
More
details
concerning
the
preparation
of
Vicia
faba
samples
are
reported
in
a
previous
study
[6].
Vicia faba samples are reported in a previous study [6].
2.2.
2.2.Acquisition
AcquisitionofofFTIR
FTIRand
andFTNIR
FTNIRSpectra
SpectraofofVicia
ViciaFaba
FabaSamples
Samples
FTIR
FTIR and
and FTNIR
FTNIR spectra
spectra were
were collected
collected for
for the
the same
same subsamples
subsamples previously
previously described.
described. FTIR
FTIR
spectra
spectrawere
werecollected
collectedby
byaaNicolet
Nicoletsingle
singlebeam
beamspectrophotometer
spectrophotometermod.
mod.IS50
IS50(Waltham,
(Waltham,MA,
MA,USA),
USA),
equipped
crystal accessory.
accessory. The
Thespectrum
spectrumofofthe
thediamond
diamondcrystal
crystalwas
was
considered
equipped with
with aa diamond
diamond crystal
considered
as
as
spectroscopic
background.
For
Vicia
faba
samples,
the
lyophilized
powder
of
the
samples
was
placed
spectroscopic background. For Vicia faba samples, the lyophilized powder of the samples was placed
directly
directlyon
onthe
thecrystal.
crystal.
−1 resolution,
Spectral
acquisition
performed
in the
cm−1 range
after 100
Spectral acquisitionwas
was
performed
in 650–4000
the 650–4000
cm−1 with
range4 cm
with
4 cm−1 resolution,
scans,
using
the
cosine
function
as
apodization.
Each
FTIR
spectrum
was
baseline
corrected
using
after 100 scans, using the cosine function as apodization. Each FTIR spectrum was baseline corrected
the
so called
point method
[11] available
on several
andFTIR
FTNIR
Given a
using
the soselection
called selection
point method
[11] available
onFTIR
several
andinstruments.
FTNIR instruments.
spectrum
such
as
the
plot
below
(Figure
3),
by
means
of
the
graphical
user
interface
we
selected
Given a spectrum such as the plot below (Figure 3), by means of the graphical user interface we
some
points
in points
the plotinwhere
thewhere
linearthe
curves
are
identified.
selected
some
the plot
linear
curves
are identified.
Figure3.3.Example
Exampleofofthe
theselection
selectionpoint
pointmethod
methodapplication
applicationfor
foraaspectrum
spectrumto
todraw
drawline
linecurves.
curves.
Figure
Then
areas under
underthe
thestraight
straight
lines
subtracted
the original
spectral
signals
to
Then the
the areas
lines
areare
subtracted
fromfrom
the original
spectral
signals
to obtain
obtain
the
following
baseline
drift
corrected
spectrum
(Figure
4)
.
the following baseline drift corrected spectrum (Figure 4).
J. Imaging 2017, 3, 11
J. Imaging 2017, 3, 11
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Figure 4. Example of a spectrum before (black) and after baseline drift correction (red) using the
Figure 4. Example of a spectrum before (black) and after baseline drift correction (red) using the
selection point method.
selection point method.
After
baseline
correction,
we submitted
the and
FTIRFTNIR
and FTNIR
an 11
point
After
baseline
driftdrift
correction,
we submitted
the FTIR
spectraspectra
to an 11topoint
smoothing
smoothing
filter
to reduce
noise
and then
the signal-to-noise
ratio according
to thesmoothing
efficient
filter
to reduce
noise
and then
enhance
theenhance
signal-to-noise
ratio according
to the efficient
and technique
differentiation
technique
for called
analytical
signals Golay
calledfilters
the [11,12].
SavitzkiIt Golay
andsmoothing
differentiation
for analytical
signals
the Savitzki
is based
filters
[11,12].
It
is
based
on
the
convolution
of
the
original
signals
by
an
appropriate
smoothing
on the convolution of the original signals by an appropriate smoothing (i.e., polynomial) filter
function
polynomial)
filter function “f” according to
“f ”(i.e.,
according
to
j j== m
m
X’i = Σ fj X(i + j) i = 1, 2, 3 . . . . . . .,n
X’i = Σ fj X(i + j) i = 1, 2, 3…….,n
j = −m
j = −m
where X is the n-point absorption signal file, X’ is the smoothed signal file, and m is the number of the
where X is the n-point absorption signal file, X’ is the smoothed signal file, and m is the number of
filtering points. For instance, in the case of a FTIR spectrum in this study, the application of an 11 point
the filtering points. For instance, in the case of a FTIR spectrum in this study, the application of an
filter means that m = 5, so that each final X’i smoothed signal is determined by taking into account
11 point filter means that m = 5, so that each final X’i smoothed signal is determined by taking into
the previous five Xj values, the Xi value, and the next five Xj values. With respect to a conventional
account the previous five Xj values, the Xi value, and the next five Xj values. With respect to a
smoothing filter such as the moving window filter, where
conventional smoothing filter such as the moving window filter, where
m
j j== m
(1) (1)
= 1/(2m
+ 1)
i =1,1,2,2,33…….,
X’i =X’i
1/(2m
+ 1)
ΣΣ
XX
(i (i+ +j)j)i =
. . . . . .n., n
(2) (2)
j j==−−m
m
(3) (3)
thethe
presence
of the
f -function
filter
allows
a very
reliable
restoration
of the
signal
files,
avoiding
peak
presence
of the
f-function
filter
allows
a very
reliable
restoration
of the
signal
files,
avoiding
peak distortions
[11,12].two
These
two references
give practical
of this smoothing
filter
distortions
[11,12]. These
references
also givealso
practical
examplesexamples
of this smoothing
filter utilization.
utilization.
Finally,
the saved
spectraaswere
saved
ASCII
filesdigital
for further
digital elaborations.
Finally,
the spectra
were
ASCII
filesasfor
further
elaborations.
The
absence
residual
water
after
lyophilisation
was
tested
the
negligiblepresence
presenceofofthe
theflat
The
absence
of of
residual
water
after
lyophilisation
was
tested
byby
the
negligible
−1 (Figure 1). For the examination of the results
flat band,
water ranging
band, ranging
between
and 2500
water
between
1900 1900
and 2500
cm−1cm
(Figure
1). For the examination of the results we
we considered
the average
spectrum
of three
spectral
subsamples
each
concentration.
considered
the average
spectrum
of three
spectral
subsamples
for for
each
As As
concentration.
FTNIR
spectra
were
collected
in
diffuse
reflectance
(DRIFT)
mode
using
beam
FTNIR spectra were collected in diffuse reflectance (DRIFT) mode usinga aJasco
Jascosingle
single
beam
spectrophotometer
mod.
420
(Jasco
Europe
Srl,
Milano,
Italy),
equipped
with
an
EasyDiff
(Pike
spectrophotometer mod. 420 (Jasco Europe Srl, Milano, Italy), equipped with an EasyDiff (Pike
Technology, Madison, WI, USA) accessory. The powder of lyophilized Vicia faba samples (between
Technology,
Madison, WI, USA) accessory. The powder of lyophilized Vicia faba samples (between
10 and 20 mg) were placed in a steel cup. The spectra were collected in the 10,000 to 4000 cm−1 range−1
10 and 20 mg) were placed in a steel cup. The spectra were collected in the 10,000 to 4000 cm
J. Imaging 2017, 3, 11
6 of 13
range at spectral resolution 4 cm−1 after 500 scans, using the cosine function as apodization. For
FTNIR
measurements,
ultrapure dried KBr was used for spectral background. Similar to 6the
J. Imaging
2017, 3, 11
of 13FTIR
spectra, FTNIR spectra were baseline corrected, submitted to an 11 point smoothing filter and like FTIR
−1 after 500 scans, using the cosine function as apodization. For FTNIR
at spectralthe
resolution
cmthree
spectroscopy,
average4 of
replicated spectral subsamples were considered. FTNIR spectra
measurements,
ultrapure
dried
KBr was used for spectral background. Similar to the FTIR spectra,
were also saved as ASCII files.
FTNIR spectra were baseline corrected, submitted to an 11 point smoothing filter and like FTIR
spectroscopy,
three replicated
spectral
subsamples
were
considered. FTNIR spectra
2.3. Basic
Theory of the
the average
ProposedofDigital
Examination
of FTIR
and FTNIR
Spectra
were also saved as ASCII files.
2.3.1. The Examination of Digitised FTIR Spectra
2.3. Basic Theory of the Proposed Digital Examination of FTIR and FTNIR Spectra
The digital approach for spectral examinations consists of a routine in the Matlab (Mathworks Inc.,
The Examination
of Digitised
FTIR spectra
Spectra saved as ASCII files. The Matlab routine performs
Natik,2.3.1.
IL, USA)
language which
retrieves
the examination
of FTIR
spectra
four separated
steps.
In the
step,inthe
the exposed
The digital
approach
for in
spectral
examinations
consists
of first
a routine
thecontrol
Matlaband
(Mathworks
spectral
areUSA)
normalized
to make
independent
of the
of sample
used routine
for spectral
Inc.,samples
Natik, IL,
language
which them
retrieves
spectra saved
as amount
ASCII files.
The Matlab
acquisition
[11].
The
applied
normalization
method
works
on
the
n-y
series
of
signal
intensities
of
performs the examination of FTIR spectra in four separated steps. In the
i first step, the control and
the exposed
spectral samples
are normalized
to make
them independent
the amount
sample
each spectrum
identifying
the maximum
and the
minimum
values of theofsignal
series.ofThen
all the
used are
for re-determined
spectral acquisition
[11]. The applied
normalization
method
works on all
thethe
n-yvalues
i series by
of the
yi values
by subtracting
the minimum
value
and dividing
signal intensities
of each
spectrumand
identifying
the maximum
and the
minimum
of the signal
differences
between the
maximum
minimum
values. After
this
type of values
normalization,
all the
series. Then all the yi values are re-determined by subtracting the minimum value and dividing all
spectra (FTIR and FTNIR) have absorptions ranging between 0 and 1.
the values by the differences between the maximum and minimum values. After this type of
In the second step, the control and exposed samples are preliminarily plotted jointly to evidence
normalization, all the spectra (FTIR and FTNIR) have absorptions ranging between 0 and 1.
the spectral
difference
andexposed
structural
changes)
in the same plotted
map (please
In the
second (i.e.,
step, quantitative
the control and
samples
are preliminarily
jointly see
to the
example
of
FTIR
spectra
in
Figure
1).
evidence the spectral difference (i.e., quantitative and structural changes) in the same map (please
In
step,
the spectral
ranges
seethe
thethird
example
of FTIR
spectra in
Figureof
1).DNA and RNA, Amide I and Amide II, and lipids are
plotted to In
give
detailed
of theofstructural
caused
by Amide
As (Figure
5).lipids
The spectral
theathird
step, visualization
the spectral ranges
DNA and changes
RNA, Amide
I and
II, and
are
plotted
to give a the
detailed
visualization
of of
theall
structural
changes caused
by of
AsDNA,
(FigureRNA,
5). The
ranges
for detecting
structural
changes
of the molecular
groups
lipids,
spectral
ranges
for
detecting
the
structural
changes
of
all
of
the
molecular
groups
of
DNA,
RNA,
carbohydrates, and proteins can be retrieved by means of the most significant FTIR (Table 1).
lipids, carbohydrates, and proteins can be retrieved by means of the most significant FTIR (Table 1).
Figure
5. Visualization
ofcomparison
the comparison
between
control
(black)
exposed
(red)
Figure
5. Visualization
of the
between
the the
control
(black)
andand
exposed
(red)
AsAs
(10(10
g·L−1 )
g·L−1) sample in the FTIR spectral range corresponding to the absorptions of the DNA and RNA
sample in the FTIR spectral range corresponding to the absorptions of the DNA and RNA region;
region; proteins; unsaturated lipids; fatty acids; and lipid disorder. The spectral ranges are reported
proteins; unsaturated lipids; fatty acids; and lipid disorder. The spectral ranges are reported starting
starting from the top to the bottom plot.
from the top to the bottom plot.
In the fourth step, by means of the digitized examination of the FTIR spectra we determine the
four
to detect examination
structural changes
arising
the we
As determine
treatment. the
In thefundamental
fourth step,parameters
by meansselected
of the digitized
of the
FTIR from
spectra
These parameters
are the selected
DNA and
RNA area
ratio (DNA-RNA),
protein
(PO),
four fundamental
parameters
to detect
structural
changes arising
fromoxidation
the As treatment.
J. Imaging 2017, 3, 11
7 of 13
These parameters are the DNA and RNA area ratio (DNA-RNA), protein oxidation (PO), unsaturated
to saturated lipid ratio (USL), and fatty acid area ratio (FA). All these parameters are determined
as relative percent changes of the exposed sample band vs. the control sample band area, showing
relative reduced or increased signal intensities.
Let us make a numerical example, regarding plot A of Figure 5. DNA and RNA bands are located
in the 1300 to 1000 cm−1 range. Firstly, the routine measures the integral of the spectral range, then the
two integrals (i.e., control and exposed samples) in the selected range are considered to calculate the
area ratio. The selection of the band area instead of the peak height is always suggested because it is
more representative and accurate for describing the structural properties and changes of the molecules
such as polar and non-polar interactions [5,10].
The PO parameter needs a separate evaluation because it is determined by means of the change
ratios in two different spectral ranges, the Amide II range between 1520 and 1580 cm−1 and the range
of Amide I between 1580 and 1700 cm−1 [1,5]. Protein oxidation in living cells produces the formation
of molecules called non-protein molecules, where the –C=O group has infrared absorptions, placed
at higher wavenumbers (1700 and 1750 cm−1 ) with respect to the protein –C=O group, whereas the
absorption of the Amide II band results unchanged [13]. This determines that a positive PO value (i.e.,
higher in the exposed sample than in the control sample) suggests the presence of protein oxidation
and/or depletion [5,14]. A lower or an unchanged PO value with respect to the control sample points
out a generic variation of the secondary protein structure without specifying the presence of protein
oxidation. This is the case of protein denaturation (please see Section 4.1.2.).
After uploading the control and exposed sample spectral files, the time taken by the computer to
perform the analysis is only a few seconds.
2.3.2. The Examination of Digitised FTNIR Spectra
The Matlab routine for FTNIR spectroscopy consists of three steps. The first two steps (i.e.,
normalization and plot of the whole FTNIR spectra) are the same as those described for FTIR
spectroscopy. In the third step the routine plots and measures two specific parameters (i.e., with
the control to sample ratio area). The first parameter measured by FTNIR spectroscopy is the ratio
between the control and exposed sample of the first overtone band of the methyl group in aliphatic
chains (MET1overt in the 6000 to 5600 cm−1 range. This band is the marker of specific alterations in
living cells [15]. The second parameter belongs to the combination band between 5300 and 4900 cm−1
(IntOHCONH), related to both the inter and intra molecular hydrogen bond interactions among the
Amide I and Amide II bands which characterize the secondary protein structure [8]. The spectral
ranges are selected according to Table 2, also taking into account that, as already reported, they show
the most significant spectral changes.
2.4. Chemical Reagents
All the chemicals (water, solvents, and As) were of analytical reagent grade.
3. Results
3.1. Conventional Interpretation of FTIR and FTNIR Spectra for Living Organisms
3.1.1. FTIR Spectroscopy
The absorption bands in the ranges between 3050 and 3000 cm−1 and between 2950 and
2800 cm−1 belong to the –CH and –CH2 groups of unsaturated and saturated aliphatic chains of
lipids, respectively [16,17], though there is also a contribution from the aliphatic chains of proteins.
The absorption ratio of these two ranges can be used to estimate the changes of the unsaturated to
saturated lipid ratio (USL), because it is the marker of lipid oxidation [17]. Lipid oxidation occurs when
cells are submitted to stress conditions which affect the dynamic properties of their membranes [18,19].
J. Imaging 2017, 3, 11
8 of 13
Moreover, the modified shape of the –CH2 aliphatic chains in the 2800 to 3050 cm−1 range can also
estimate the increased lipid disorder, another useful marker of lipid alterations in the membrane
dynamics [18–20].
The bands in the 1750 to 1700 cm−1 range belong to the –C=O carbonyl group of fatty and ester
fatty acids [1]. The changes of intensities and/or band shapes in this range suggest the presence of
lipid oxidation structural changes, related to the changes of the USL ratio previously described [21].
Therefore, the measurements of the ratio band intensities in the 1750 to 1700 cm−1 range between the
control and exposed samples, called FA, is an overview of the structural changes in fatty acids. In fact,
lipid oxidation, which is more reasonably a lipid peroxidation [22], produces increased carbonyl
compound contents [23] that modify the lipid to protein ratio (i.e., the LPR value). This parameter is
easily measurable by means of FTIR spectroscopy [20].
The two main bands in the 1700 to 1520 cm−1 range belong to the so-called Amide I and Amide II
bands of the HN–C=O peptidic group of proteins [1]. This spectral range has plenty of information
about protein structures [24]. By determining the second derivative of the FTIR spectra in the 1700 to
1620 cm−1 sub-range, the changes in the secondary protein structures involving alpha helix, beta sheet,
beta turn, and random coil transitions can also be detected [5,14,25–28] Moreover, the changes of the
ratio between the 1580–1520 cm−1 and the 1700–1580 cm−1 (PO) sub-ranges evidence protein depletion
and oxidation [3,14,28,29]. The fatty acid range between 1700 and 1750 cm−1 (already described),
when joined to the 1700 to 1620 cm−1 Amide I range of proteins, is also used to determine the lipid to
protein ratio (LPR) which is another marker of protein oxidation [20,21].
The bands ranging between 1250 and 1050 cm−1 belong to the –CO (i.e., backbone carbohydrates)
and P=O groups of DNA and RNA molecules and of phospholipids, respectively [2,3,6,21].
3.1.2. FTNIR Spectra
FTNIR bands are less numerous than those of FTIR spectra as shown by Figure 2 and Table 2.
However, the structural changes evidenced by FTNIR spectroscopy gives us peculiar structural
information. The MET1overt parameter measured by comparing the 6000 and 5600 cm−1 (first overtone
of aliphatic chains) absorptions of the control and exposed sample, is linked to the re-orientation of
lipids called lipid disorder [15], already described by means of FTIR spectra. Changes in hydrogen
bond interactions (i.e., the IntHCONH parameter), measured as the ratio in the 5300 to 4900 cm−1
range between the control and exposed sample, describes the interaction among –C=O and -NH groups
of proteins, linked to inter and intra hydrogen bond changes of proteins [8,9].
4. Discussion
4.1. As Effects on Vicia Faba Roots by the Proposed Computer Assisted Method
Arsenic, selected as an example for the proposed method, has a natural occurrence in the
environment, ranked as the 20th most abundant element in the earth’s crust. Weathering of rocks
converts As2 S3 to As2 O3 which enters the food chain by dissolution in groundwater [30]. Though As
also has recognized anti-cancer properties [31,32], it produces a variety of health problems such as
immunologic, genotoxic, mutagenetic, and carcinogenic effects [30]. For these reasons, its effects on
living organisms are currently under investigation.
We divide the discussion of As effects on Vicia faba roots into quantitative (reduction and increase)
changes and non-specific (i.e., transition) changes.
4.1.1. Quantitative Changes Detected by FTIR Spectroscopy in Vicia Faba Roots
The plots of Figure 6 are an example of the results obtained by FTIR spectroscopy for the two
different times of As treatment; June (top plot) and July (bottom plot). As exposure in the June samples
has a major effect on DNA-RNA changes with essentially a minor relevance for the LPR marker. All the
remaining parameters (PO, USL, and FA) show little (<10%) or even negligible quantitative changes.
J. Imaging 2017, 3, 11
9 of 13
FTIR also suggests that the effect of As is time dependent. In fact, for the June exposure (Figure 6,
upper plot), the DNA-RNA parameter has an increasing trend with a relative variation close to 50%,
J. Imaging 2017, 3, 11
9 of 13
whereas in the July exposure the DNA-RNA parameter has a decreasing trend, with close to 20%
reduction
(Figure
6, bottom
variation
close
to 50%, plot).
whereas in the July exposure the DNA-RNA parameter has a decreasing
trend, with close to 20% reduction (Figure 6, bottom plot).
% Band area variaiton
10 g / L
100
75
50
25
0
-2 5
D N A -R N A
PO
LPR
FA
US L
-5 0
20 g /L
% Band area variaition
20 g /L
30 g /L
20
10
0
-1 0
D N A -R N A
PO
LPR
FA
US L
-2 0
-3 0
Figure
Barcharts
charts of
area
variation
with with
respectrespect
to the control
the FTIR in the
Figure
6. 6.Bar
ofthe
theband
band
area
variation
to thesample
controlobserved
sampleinobserved
spectra for the As exposure of the June (upper) and July (bottom) plots. Here, the results are the average
FTIR spectra for the As exposure of the June (upper) and July (bottom) plots. Here, the results are the
of the three replicated spectra for each As exposure.
average of the three replicated spectra for each As exposure.
The LPR marker has a 20% reduction for the June exposure (upper plot) whereas for the July
it is almost
close to 5 %
theJune
July exposure
(bottom
plot).
Thewhereas
LPR variations
Theexposure
LPR marker
hasnegligible,
a 20% reduction
forforthe
exposure
(upper
plot)
for the July
observed for the June treatment suggest that the stress effect caused by As exposure produces an
exposure it is almost negligible, close to 5 % for the July exposure (bottom plot). The LPR variations
increased lipid disorder (Figure 8). These results are described in better detail in the following
observed
for the June treatment suggest that the stress effect caused by As exposure produces an
section (please see Section 3.2.2).
increased lipid
results
are described
in better
detailchanges
in the because
following
section
(please see
The disorder.
remainingThese
markers
(PO, USL,
and FA) have
negligible
their
variations
Section (increase
4.1.2). or decrease) are lower than 10%, lying in the range of the experimental error.
These results
show that
theUSL,
As effects
mainly
to produce
DNAbecause
and RNAtheir
changes
The remaining
markers
(PO,
andare
FA)
haveoriented
negligible
changes
variations
and
to
a
minor
extent,
LPR
structural
changes.
These
changes
depend
on
the
different
experimental
(increase or decrease) are lower than 10%, lying in the range of the experimental error.
conditions of As treatment in all cases (i.e., the simple comparison between plots A and B of
These results show that the As effects are mainly oriented to produce DNA and RNA changes
Figure 5). The FTIR spectra of the other samples confirm the time dependence effect of As.
and to a minor extent, LPR structural changes. These changes depend on the different experimental
conditions
ofNon
As Specific
treatment
in all cases
(i.e.,
the simple
comparison
between
plots
A and B of Figure 5).
4.1.2.
Structural
Changes
Detected
by FTIR
Spectroscopy
in Vicia faba
Roots
The FTIR spectra
of
the
other
samples
confirm
the
time
dependence
effect
of
As.
Figure 7 reports the FTIR spectral range of the Amide I band in derivative mode for the As
exposure in June, highly comparable with those in July, showing that the transition changes caused
4.1.2. Non
Specific
Structural
Changes
Detected
by environmental
FTIR Spectroscopy
inconditions.
Vicia faba Roots
by As
in proteins
are generally
independent
of the
and time
In addition, As exposure also caused some molecular re-arrangements involving the two alpha
Figure 7 reports the FTIR spectral range of the
Amide I band in derivative mode for the As
helix bands, which are located at 1646 and 1657 cm−1 for the control sample, whereas in the treated
exposure
in
June,
highly
comparable
with
those
in
July,
showing that the transition changes caused by
sample we observed only one alpha band, shifted at 1652 cm−1. Since the protein oxidation reactions
As in proteins
are generally
independent
the
environmental
andthan
time
conditions.
were almost
negligible, as
shown by theof
PO
parameter
values lower
10%
(Figure 6, both plots),
In addition,
As exposure
caused
some
re-arrangements
involving
protein denaturation
and also
unfolding
effects
aremolecular
more relevant
than oxidative effects.
This the
lets two
us alpha
−1 for thecause
hypothesize
that located
arsenic atexposure
essentially
thesample,
partially
formation
of treated
helix bands,
which are
1646 andcould
1657 cm
control
whereas
in the
−
1
sample we observed only one alpha band, shifted at 1652 cm . Since the protein oxidation reactions
were almost negligible, as shown by the PO parameter values lower than 10% (Figure 6, both plots),
protein denaturation and unfolding effects are more relevant than oxidative effects. This lets us
J. Imaging 2017, 3, 11
10 of 13
hypothesize
that3,arsenic
exposure could essentially cause the partially formation of oligopeptide-metal
J. Imaging 2017,
11
10 of 13
complexes, according to a mechanism of detoxication already observed in vegetal and animal
J. Imaging 2017, 3, 11
10 of 13
oligopeptide-metal
complexes,
to asuch
mechanism
ofPb
detoxication
already observed in vegetal
organisms
for As [33,34]
and otheraccording
pollutants
Cd and
[35].
and animal organisms for As [33,34] and other pollutants such Cd and Pb [35].
oligopeptide-metal complexes, according to a mechanism of detoxication already observed in vegetal
and animal organisms for As [33,34] and other pollutants such Cd and Pb [35].
Figure 7. Second derivative FTIR spectra of Amide I bands in Vicia faba roots for the As exposure in June
Figure 7. Second derivative FTIR spectra of Amide I bands in Vicia faba roots for the As exposure in
for the same sample of Figure 6. For the July exposure, the FTIR spectra are highly comparable. The
June for the same sample of Figure 6. For the July exposure, the FTIR spectra are highly comparable.
Figure
7. Second
FTIR spectra
of Amide
I bands inisVicia
faba rootssample.
for the As
exposure
blackThe
spectrum
is the derivative
control samples,
and the
red spectrum
the exposed
The
arrowsinshow
black spectrum is the control samples, and the red spectrum is the exposed sample. The arrows
−
1
−1
June
for
the
same
sample
of
Figure
6.
For
the
July
exposure,
the
FTIR
spectra
are
highly
comparable.
the transitions
between between
the betathe
sheet
with
the appearance
of random
coil
(1637
−1) with
show the transitions
beta(1632
sheetcm
(1632) cm
the appearance
of random
coil
(1637cm )
−
1
The
black
spectrum
is
the
control
samples,
and
the
red
spectrum
is
the
exposed
sample.
The
arrows
and molecular
rearrangements
within within
the alpha
helix helix
structures
in the
1645
to 1660
cmcm−1 range.
rearrangements
the alpha
structures
in the
1645
to 1660
range.
cm−1) and molecular
show the transitions between the beta sheet (1632 cm−1) with the appearance of random coil (1637
cm−1) and molecular rearrangements within the alpha helix structures in the 1645 to 1660 cm−1 range.
non-specific
effects
Asconsist
consist of
of aa denaturation
denaturation ofofproteins
caused
by hydrogen
bondbond
The The
non-specific
effects
ofofAs
proteins
caused
by hydrogen
transitions and protein unfolding, involving the Amide I and Amide II bands of proteins [25–29].
transitionsThe
and
protein unfolding,
involving
Amide I and
Amide caused
II bands
proteins
[25–29].
non-specific
effects of As
consist of the
a denaturation
of proteins
by of
hydrogen
bond
As shown in Figure 7, this transition/denaturation is clearly linked to the modification of the
transitions
and protein
unfolding, involving the Amide
I and
Amide
II bands
of proteins
As shown
in Figure
7, this transition/denaturation
is clearly
linked
to the
modification
of[25–29].
the β-sheet
β-sheet band at 1632 cm−1 present in the control sample and absent in the exposed sample. In fact, in
1 present
As
showncm
in−Figure
7, this
transition/denaturation
is absent
clearly linked
to
the modification
of
bandthe
at exposed
1632
in
the
control
sample
and
in
the
exposed
sample.
In the
fact,
in
samples we−1 observe the presence of a new band, the random coil band, present
at
β-sheet
band
at
1632
cm
present
in
the
control
sample
and
absent
in
the
exposed
sample.
In
fact,
in
the exposed
we observe the presence of a new band, the random coil band, present at
1637 cm−1samples
[25].
the−exposed
1 [25]. samples we observe the presence of a new band, the random coil band, present at
1637 cm
Concerning
lipid
disorder, Figure 8 shows analogous modified shapes of the band placed
1637 cm−1 [25].
between
3000lipid
and 2800
cm−1 related
the transitions
between
the trans
andofgauche
conformations
Concerning
disorder,
Figure 8toshows
analogous
modified
shapes
the band
placed between
Concerning
lipid disorder, Figure 8 shows analogous modified shapes of the band placed
−
1
of
the
aliphatic
(i.e.,
lipid)
chains
of
the
–CH
2
acyl
bands
[36,37].
3000 and
2800 cm related to the transitions between the trans and gauche conformations of the
between 3000 and 2800 cm−1 related to the transitions between the trans and gauche conformations
aliphatic
(i.e.,
lipid)(i.e.,
chains
of chains
the –CH
bands
[36,37].
2 acyl
of the aliphatic
lipid)
of the
–CH
2 acyl bands [36,37].
Figure 8. FTIR spectra in second derivative mode showing the increased lipid disorder by the band
shifts of the lipid acyl –CH2 bands in samples exposed to As in June and July. This FTIR plot
Figure
8. FTIR
spectra
in second
derivative
theincreased
increased
lipid
disorder
by band
the band
Figure
8. FTIR
spectra
in second
derivativemode
mode showing
showing the
lipid
disorder
by the
includes all the samples considered in the study. As a qualitative case, the average and standardized
shifts
the acyl
lipid–CH
acyl2 –CH
2 bands
in samples
exposed
toinAs
in June
and July.
This
FTIR
plot
shiftsspectrum
of theoflipid
bands
in
samples
exposed
to
As
June
and
July.
This
FTIR
plot
includes
of each sample was considered. “J10” and “J20” are June samples treated with As 10 and
includes
allconsidered
the samplesin
considered
inAs
theastudy.
As a qualitative
case,
the average
and standardized
all the
samples
the
study.
qualitative
case,
the
average
and
standardized
spectrum
−1
20 g·L , respectively. “JL10” and “JL20” are July samples treated with As 10 and 30 g·L−1,
spectrum
each sample was considered.
“J10” and
“J20” samples
are June samples
treated
with
1020
and
of each
sampleofwas
and colour
“J20”
are
June
treated
with where
As
10 As
and
g ·L−1 ,
respectively.
The considered.
arrows (with“J10”
the same
of the
sample) show
the ranges
the band
101 and 30 g·L−1,
20 g·L−1, respectively. “JL10” and “JL20” are July samples treated with As −
respectively.
“JL10”
“JL20”
arewith
Julyrespect
samples
treated
As 10 and 30 g·L , respectively. The
shape changes
areand
more
relevant
to the
controlwith
samples.
respectively. The arrows (with the same colour of the sample) show the ranges where the band
arrows (with the same colour of the sample) show the ranges where the band shape changes are more
shape changes are more relevant with respect to the control samples.
relevant with respect to the control samples.
Figure 6. The results confirm the time dependent effect, already shown by the FTIR results. For the
June samples, we observed the reduction of the IntOHCONH parameter which is due to the
combination bands (i.e., hydrogen bonds) among the proteins and polysaccharides (Table 2). These
reduction-changes, about 15%, agree with the almost significant variation of the PO parameter
measured by FTIR spectroscopy (Figure 6) and with the structural changes, linked to protein
denaturation,
J. Imaging
2017, 3, 11 detected by the second derivative FTIR spectroscopy (Figure 7).
11 of 13
The Met1overt parameter measured in the 6000 to 5600 cm−1 range belongs to the first overtone
bands of the –CH3 and –CH2 groups of aliphatic chains in lipid compounds (Table 2). This change is
4.1.3.significant
FTNIR Results
(roughly 50%), and agrees with the lipid disorder already observed by the FTIR
spectroscopy results shown in Figure 5E, and in more detail with the acyl bands of Figure 8
Figure 9 reports the values of IntHCONH and Met1overt for the same samples shown in Figure 6.
describing the lipid disorder induced by the As exposure.
The results
confirm
thesamples,
time dependent
effect, already
by theincrease
FTIR results.
samples,
For
the July
the IntOHCONH
value shown
had a great
(FigureFor
9). the
ThisJune
finding
we observed
the reduction
the IntOHCONH
parameter
which
is due to the
combination
bands (i.e.,
agrees with
the proteinofdenaturation
detected
by the FTIR
spectroscopy
showing
the transitions
hydrogen
bonds)
amongand
thethe
proteins
(Table 2).
These
between
the β-sheet
randomand
coil polysaccharides
structure re-arrangement
among
thereduction-changes,
alpha helix structuresabout
7). The
parametervariation
related toofthe
contents by
shows
negative
15%, (Figure
agree with
theMet1overt
almost significant
thelipid
PO structures
parameterand
measured
FTIRa spectroscopy
change,
close
to
50%,
comparable
with
those
observed
for
the
June
samples.
This
result
shows
(Figure 6) and with the structural changes, linked to protein denaturation, detected by thethat
second
FTNIRFTIR
spectroscopy
can support
derivative
spectroscopy
(Figurethe
7).evidence of lipid disorder effects already shown by the FTIR
spectra of Figure 8.
% Band area variaiton
10 g /L
0
-1 0
M e t1 o v e r t
I n tO H C O N H
-2 0
-3 0
-4 0
-5 0
-6 0
20 g /L
% Band area variaition
20 g /L
600
500
400
300
200
100
0
-1 0 0
-2 0 0
M e t1 o v e r t
30 g /L
I n tO H C O N H
Figure
9. Bar
charts
of the
bandarea
areavariation
variation with
with respect
sample
observed
in thein the
Figure
9. Bar
charts
of the
band
respecttotothe
thecontrol
control
sample
observed
FTNIR
spectra
for As
the exposure
As exposure
of the
June
(upper)and
andJuly
July(bottom)
(bottom) plots.
plots. Here,
FTNIR
spectra
for the
of the
June
(upper)
Here, the
theresults
resultsare
are the
the
average
of
the
three
replicated
spectra
for
each
As
exposure.
average of the three replicated spectra for each As exposure.
5. Conclusions
The Met1overt parameter measured in the 6000 to 5600 cm−1 range belongs to the first overtone
The proposed method based on the computer assisted examination of FTIR and FTNIR spectra
bands of the –CH3 and –CH2 groups of aliphatic chains in lipid compounds (Table 2). This change is
allows the easy and fast investigation of molecular changes and damages caused in living cells by
significant (roughly 50%), and agrees with the lipid disorder already observed by the FTIR spectroscopy
pollutant exposures. By examining specific cell parameters in the FTIR spectra, molecular
results
shown in Figure
5, DNA,
and inRNA,
more
detail
thestructures
acyl bands
of Figure
8 describing
the lipid
modifications
related to
lipid,
andwith
protein
in Vicia
faba roots
can be detected.
disorder
induced
by
the
As
exposure.
In our opinion, this is a peculiar effort to assess specific molecular damages caused by pollutant
For
the July
samples,with
the IntOHCONH
value hadFTNIR
a greatspectroscopy
increase (Figure
9).able
Thistofinding
agrees
exposure.
However,
a reduced contribution,
is also
provide
with the protein denaturation detected by the FTIR spectroscopy showing the transitions between the
β-sheet and the random coil structure re-arrangement among the alpha helix structures (Figure 7).
The Met1overt parameter related to the lipid structures and contents shows a negative change, close to
50%, comparable with those observed for the June samples. This result shows that FTNIR spectroscopy
can support the evidence of lipid disorder effects already shown by the FTIR spectra of Figure 8.
5. Conclusions
The proposed method based on the computer assisted examination of FTIR and FTNIR spectra
allows the easy and fast investigation of molecular changes and damages caused in living cells by
pollutant exposures. By examining specific cell parameters in the FTIR spectra, molecular modifications
related to DNA, RNA, lipid, and protein structures in Vicia faba roots can be detected. In our opinion,
this is a peculiar effort to assess specific molecular damages caused by pollutant exposure. However,
J. Imaging 2017, 3, 11
12 of 13
with a reduced contribution, FTNIR spectroscopy is also able to provide supporting data regarding
molecular damage in living cells, related to protein denaturation and lipid disorder in Vicia faba roots.
Due to its performance, the proposed method for the examination of digitised vibrational spectra
can be applied jointly to the common tests of the acute toxicity of pollutants, contributing to a wider
knowledge of their effects on living organisms.
Author Contributions: Mauro Mecozzi and Elena Sturchio conceived and designed the study and the experiments
jointly. Elena Sturchio took care of the field experiments related to As exposure of Vicia faba and then to
their harvest and preparation for spectroscopic (FTIR and FTNIR) analysis. Mauro Mecozzi performed the
spectroscopic measurements and developed the computational routines for spectral analysis. The results were
discussed together.
Conflicts of Interest: The authors declare no conflict of interest.
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