sensors
Article
In-Field, In Situ, and In Vivo 3-Dimensional
Elemental Mapping for Plant Tissue and Soil Analysis
Using Laser-Induced Breakdown Spectroscopy
Chunjiang Zhao, Daming Dong *, Xiaofan Du and Wengang Zheng
National Engineering Research Center for Information Technology in Agriculture,
Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China;
[email protected] (C.Z.); [email protected] (X.D.); [email protected] (W.Z.)
* Correspondence: [email protected]; Tel.: +86-10-5150-3654
Academic Editor: Russell Binions
Received: 28 July 2016; Accepted: 19 October 2016; Published: 22 October 2016
Abstract: Sensing and mapping element distributions in plant tissues and its growth environment has
great significance for understanding the uptake, transport, and accumulation of nutrients and harmful
elements in plants, as well as for understanding interactions between plants and the environment.
In this study, we developed a 3-dimensional elemental mapping system based on laser-induced
breakdown spectroscopy that can be deployed in- field to directly measure the distribution of multiple
elements in living plants as well as in the soil. Mapping is performed by a fast scanning laser, which
ablates a micro volume of a sample to form a plasma. The presence and concentration of specific
elements are calculated using the atomic, ionic, and molecular spectral characteristics of the plasma
emission spectra. Furthermore, we mapped the pesticide residues in maize leaves after spraying to
demonstrate the capacity of this method for trace elemental mapping. We also used the system to
quantitatively detect the element concentrations in soil, which can be used to further understand the
element transport between plants and soil. We demonstrate that this method has great potential for
elemental mapping in plant tissues and soil with the advantages of 3-dimensional and multi-elemental
mapping, in situ and in vivo measurement, flexible use, and low cost.
Keywords: in-field; in situ; in vivo; elemental mapping; laser-induced breakdown spectroscopy
1. Introduction
Sensing and imaging elemental concentrations in biological tissues has great importance for
understanding uptake, transport, and accumulation of both nutritious and harmful elements in plants
as well as cell absorption and formation processes [1]. These elements include macro nutrient elements,
which are the major elements that compose tissues; micro nutrient elements; and toxic elements. In the
past few decades, scientists have continuously researched novel elemental imaging and mapping
methods for biological tissues [1–6]. Rapid, in situ, high-resolution, and sensitive imaging are the main
trends being explored among elemental imaging methods.
Currently, three types of elemental imaging and mapping methods are used for biological tissues:
X-ray microscopy-based methods [2,7,8], mass spectroscopy-based methods [2,9–12] and fluorescent
probes [13,14]. For the X-ray microscopy methods, the commonly used methods are scanning or
transmission electron microscopy with energy-dispersive X-ray analysis (SEM-EDX or TEM-EDX) and
proton/particle-induced X-ray emission (PIXE) [2,7]. The former requires a vacuum sampling system,
but the latter can only analyze elements lighter than Na. Upon development and establishment of
synchrotron radiation (SR) systems, certain researchers have used X-ray fluorescence spectroscopy
based on synchrotron radiation (SRXRF) and X-ray absorption spectroscopy based on synchrotron
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radiation (SRXAS) to image elements in plant tissues through the SR beam-line [8,15–17]. The SR
beam-line provides a detection limit of ppm (Parts-per-million) and a space resolution near 100 nm, but
one difficultly is access to a SR beam-line [18]. Secondary ionization mass spectrometry (SIMS) is the
most-used mass spectroscopy method for elemental imaging in biological tissues [2,9,19]. Nano-SIMS
was developed in recent years which provides much higher spatial resolution than common SIMS [2].
However, SIMS requires high vacuum conditions, and a quantitative analysis is difficult due to strong
matrix effects. Certain scholars have used laser ablation inductively coupled plasma mass spectrometry
(LA-ICP-MS) and laser micro-dissection inductively coupled plasma mass spectrometry (LMD-ICP-MS)
for elemental compositions in solid surfaces [10,11,20–23], but these methods provide only a few
applications for plant tissue. One potential basis for the limited application is the dehydration effects
on the samples during the laser ablation processes, which render the plant sample unable to maintain
moisture and an intact structure.
Notably, the above methods depend on complex and expensive experimental instruments and
systems and can only be performed in a laboratory. In certain plant research fields, scientists hope
to map elements in plant tissues in-field and in vivo, which may lead to a better understanding of
the transport and accumulation laws governing nutrient and toxic elements in plants. However, the
aforementioned methods cannot provide such imaging and mapping. Consider imaging pesticide
distribution in plant tissues as an example, which we will discuss below. Mapping and quantitative
observation of pesticide residues in plants is important for studies on absorption, transport, and
accumulation of pesticides in plants. However, only a few studies used matrix-assisted laser
desorption/ionization (MALDI) mass spectrometry to image the pesticide on leaf surfaces [24–26],
which requires a sample of the leaves first and then a spray of the matrix solution. Therefore, it is
impossible to produce in situ measurements.
Therefore, an in situ and in vivo micro-imaging and mapping method for both high-concentration
and trace elements in plant tissues is needed which can be used in-field. Laser-induced breakdown
spectroscopy (LIBS) is a fast-developing spectroscopy method that analyzes elemental compositions and
concentrations through inducing the measured objects into plasma using a high-energy laser, and then
collects the emission spectra [27,28]. LIBS has been used to qualitatively and quantitatively measure
solids, liquids, and gases based on atomic, ionic, and molecular spectral characteristics [28–31]. LIBS has
also been used for fast mapping and imaging elemental distributions on solid surfaces [27,32–39].
Compared with other imaging and mapping methods, LIBS is advantageous due to its simple structure,
flexibility in use, and low cost, and it can be used in-field, is removable, and can even be used as a
handheld device [28,40]. In this study, we developed a removable elemental mapping system based on
micro-LIBS, LipsImag. We demonstrated quantitative elemental mapping through LipsImag in plant
tissues using maize leaves after a pesticide spray as an example, and we also used LipsImag to detect
the element concentrations in soil. To the best of our knowledge, this is the first method and system for
in situ and in vivo 3-dimensional elemental mapping of biological tissues that is removable and can be
used in-field.
2. Experimental Methods
2.1. LipsImag
The principles of LipsImag are as follows: (1) a laser beam from a laser generator emits to the
sample, focuses on its surface and induces a micro area into plasma status; (2) the emitted plasma
spectrum is detected by a spectrometer; (3) the presence and concentrations of some objective elements
are calculated using the spectral characteristics; (4) the focusing area of the laser beam moves to the
next point using a 3-dimensional movement platform and repeat the above steps until all of the points
in the scanning area of the first plane are measured; (5) the height of the movement platform is lowered
to the next plane and the above steps are repeated until all the planes are scanned or the sample is
penetrated. The schematic of the LipsImag apparatus is shown in Figure 1. In our study, we used
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a CFR200 laser (Quantel Ltd., Les Ulis, France) to output a 1064 nm laser beam with a maximum
we used a CFR200 laser (Quantel Ltd., Les Ulis, France) to output a 1064 nm laser beam with a
energy of 200 mJ. A sevenbands spectrometer, HR2000+ (Ocean Optics, Dunedin, FL, USA), was used
maximum energy of 200 mJ. A sevenbands spectrometer, HR2000+ (Ocean Optics, Dunedin, FL,
to collect
spectra
a resolution
of a0.2
nm andofa 0.2
spectral
band
of 200–850
USA),the
wasemitted
used to plasma
collect the
emittedwith
plasma
spectra with
resolution
nm and
a spectral
band nm.
We tested
the
spectrometer
in-field,
and
validated
its
SNR
(signal-to-noise
ratio)
as
approximately
of 200–850 nm. We tested the spectrometer in-field, and validated its SNR (signal-to-noise ratio) as
170.94.
To perform 170.94.
3-dimensional
scanning,
a precision
3-dimensional
stage, LX-8375
(Felles
Photonic
approximately
To perform
3-dimensional
scanning,
a precision 3-dimensional
stage,
LX-8375
(Felles Photonic
Instruments,
Shanghai,
waswith
usedan
in accuracy
LipsImag of
with
an accuracy
of the
7 μm.
Instruments,
Shanghai,
China), was
used inChina),
LipsImag
7 µm.
To observe
surface
observe
of height
the sample
to adjustplatform,
the height
of the movement
platform,
a
of theTosample
andthe
to surface
adjust the
of theand
movement
a Grasshopper3
(LUSTER
LightTech,
Grasshopper3
(LUSTER
LightTech,
Beijing,
China)
camera
was
used
which
was
coaxial
with
the
Beijing, China) camera was used which was coaxial with the laser focusing system. We also designed
laser focusing
We all
alsoofdesigned
a removable
stand as
to equip
allaofcomputer
the components
above,
as laser
a removable
standsystem.
to equip
the components
above,
well as
to control
the
well as a computer to control the laser shot, spectral collection, and platform movement, and to
shot, spectral collection, and platform movement, and to calculate the mapping results automatically.
calculate the mapping results automatically. The control and calculation software of LipsImag was
The control
and calculation software of LipsImag was developed in the VS2010 platform.
developed in the VS2010 platform.
Figure 1. Schematic of the LipsImag apparatus and its application for in situ and in vivo elemental
Figure 1. Schematic of the LipsImag apparatus and its application for in situ and in vivo elemental
mapping of maize.
mapping of maize.
2.2. Samples
2.2. Samples
As shown in Figure 2, the maize plants used in our study were grown naturally in the
experimental
field of 2,
the
of Agriculture
and
Forestry
(Beijing,
China).
living
As shown in Figure
theBeijing
maizeAcademy
plants used
in our study
were
grown
naturally
in theThe
experimental
plants
were
directly
used
in
the
experiment.
We
also
used
small
holly
(Ilex
chinensis
Sims)
leaves
to were
field of the Beijing Academy of Agriculture and Forestry (Beijing, China). The living plants
demonstrate the ability of LipsImag for nutrient elemental mapping, which was also grown in the
directly used in the experiment. We also used small holly (Ilex chinensis Sims) leaves to demonstrate
Beijing Academy of Agriculture and Forestry. Chlorpyrifos (C9H11Cl3NO3PS) emulsifiable concentrate
the ability of LipsImag for nutrient elemental mapping, which was also grown in the Beijing Academy
(EC) that we used in the experiment was produced by Dow AgroSciences Ltd. (Indianapolis, IN,
of Agriculture
Forestry. of
Chlorpyrifos
(C9 Hchlorpyrifos
emulsifiable
concentrate
that we
11 Cl3 NO3 PS)
USA) with aand
concentration
40.7%. To prepare
with
different concentrations,
we(EC)
diluted
usedthe
in pesticide
the experiment
was produced
Dow
AgroSciences
Ltd. (Indianapolis,
with a
various times
using water.by
The
commonly
used concentration
in our studyIN,
wasUSA)
100 mL
2
concentration
of
40.7%.
To
prepare
chlorpyrifos
with
different
concentrations,
we
diluted
the
pesticide
chlorpyrifos EC (40.7%) diluted by 50 L of water and sprayed in a 1 mu (666.7 m ) maize field. We also
various
times
The commonly
used
concentration
study The
wasspray
100 mL
used
someusing
lower water.
concentrations
to study the
detection
sensitivityinofour
LipsImag.
waschlorpyrifos
carried
out using
a 5372
Ltd.,sprayed
Shanghai,
guidelines
of the
Chinese
EC (40.7%)
diluted
bysprayer
50 L of(Worth
water and
in China)
a 1 mufollowing
(666.7 m2the
) maize
field. We
also
used some
The soil
samples
obtained
from farmland
in the Haidian
District
(Beijing,
China).
lowergovernment.
concentrations
to study
thewere
detection
sensitivity
of LipsImag.
The spray
was
carried
out using
a 5372 sprayer (Worth Ltd., Shanghai, China) following the guidelines of the Chinese government.
The soil samples were obtained from farmland in the Haidian District (Beijing, China). To avoid
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splashing, each soil sample was pressed into a tablet (diameter of 13 mm, 1 mm thick) under the
To avoid splashing, each soil sample was pressed into a tablet (diameter of 13 mm, 1 mm thick)
pressure
of 10 T with a tablet machine (FW-4A, Xin Tian Guang Ltd., Tianjin, China). In this study,
under the pressure of 10 T with a tablet machine (FW-4A, Xin Tian Guang Ltd., Tianjin, China).
60 groups of samples were prepared. The real values of element contents were tested by the Beijing
In this study, 60 groups of samples were prepared. The real values of element contents were tested
Research
Centre for Agriculture and Testing.
by the Beijing Research Centre for Agriculture and Testing.
Figure
2. The
photographs
a realapplication
application of
of LipsImag
LipsImag for
measurement.
(a) Maize
Figure
2. The
photographs
in in
a real
forananin-field
in-field
measurement.
(a) Maize
plants
for
the
elemental
mapping
experiment;
(b)
Photograph
of
a
maize
leaf
after
a
20
×
20
plants for the elemental mapping experiment; (b) Photograph of a maize leaf after a 20 ×scanning
20 scanning
by LipsImag; (c) The microstructure of a single shot on a maize leaf by LipsImag; (d) A plasma
by LipsImag; (c) The microstructure of a single shot on a maize leaf by LipsImag; (d) A plasma emission
emission spectra from the scanning that is shown in (b).
spectra from the scanning that is shown in (b).
2.3. Experiments
2.3. Experiments
The field experiment was carried out by moving LipsImag outdoor, stretching the living maize
leaves
ontoexperiment
the platform,
fixing
them out
withby
double-faced
adhesive outdoor,
tape and then
scanning.
hollymaize
The
field
was
carried
moving LipsImag
stretching
theThe
living
leaves
cannot
be
stretched
due
to
a
distance
problem
and
thus
were
cut
and
then
placed
on
theholly
leaves onto the platform, fixing them with double-faced adhesive tape and then scanning. The
platform
to
complete
the
scanning.
For
the
calibration
experiment,
a
20
μL
pesticide
sample
which
leaves cannot be stretched due to a distance problem and thus were cut and then placed on the platform
was diluted
several different
times,
from 400experiment,
to 40,000, were
onto the
surface
of thewas
maize
to complete
the scanning.
For the
calibration
a 20dropped
µL pesticide
sample
which
diluted
leaf in an area of about 1 cm2. In this way, we obtained the samples with pesticide residues of
several different2 times, from 400 to 40,000, were dropped onto the surface of the maize leaf in an area
0–30 μg/cm . Then, we used LipsImag to measure the intensities of the spectral characteristics and
of about 1 cm2 . In this way, we obtained the samples with pesticide residues of 0–30 µg/cm2 . Then,
we used LipsImag to measure the intensities of the spectral characteristics and establish a calibration
model. In this study, we used a 90 mJ laser energy, a 75 µm laser spot size and a 200–850 nm spectral
band in most cases.
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2.4. Spectral Processing and Mapping Methods
The spectral processing algorithms were embedded in the software of LipsImag. The spectra were
processed by the following steps: (1) the background line was subtracted from the plasma emission
spectra [28]; (2) the spectrum was smoothed [28]; (3) each spectra band was area-normalized in a 20 nm
window to decrease the influence of the laser jitter [40]; (4) the area of each spectral characteristics
peak was calculated as a variable for calibration [28]. After scanning, the presence and concentrations
of the specific elements of each point in the 3-dimensional scanning array had been calculated, then,
2D surface images, 3D images, and 2D cross-sectional images that show the distribution of the elements
in plant tissues were drawn.
3. Results and Discussion
3.1. In Situ and In Vivo 3-Dimensional Elemental Mapping of Plant Leaves
LipsImag consists of an LIBS module, a 3-dimensional movement platform, an observation
camera, and a removable stand, as shown in Figure 1. The measurements are generated by the
LIBS module as follows: An Nd:YAG laser outputs a 1064 nm laser pulse onto the micro area of
the measured object. The micro area will then be induced into a plasma, the plasma signal will
be collected by a fiber and dispersed by gratings, an emission spectrum will be acquired, and,
finally, the element concentrations will be calculated. The observation camera is used to observe
the sample surface micro-structure and is coaxial with the laser. Figure 1 shows how LipsImag
measures maize leaves in-field. We stretched a living leaf onto the 3-dimensional movement platform
and fixed it with double-faced adhesive tape. Next, the distance between the sample and laser was
adjusted using the observation camera. The spot size and scanning areas can be selected using
the LipsImag operating software. During the measurement, the first micro point in the first plane
was hit by the laser, and the spectrum was measured; next, each point in the scanning area was hit
and measured one-by-one through automatically adjusting the 3-dimensional movement platform.
When scanning for the first plane was completed, the height of the 3-dimensional platform was
automatically adjusted, and LipsImag began scanning the next plane. Thus, a collection of spectra that
correspond to a 3-dimensional micro area can be acquired, and a 3-dimensional elemental distribution
can be imaged. Certain leaves and stalks must be cut and then placed onto the platform if they are
unable to be stretched. Notably, the LipsImag is much simpler than mass spectroscopy-based and
X-ray microscopy-based imaging and mapping systems.
The photographs that we collected using LipsImag for field measurements of maize leaves are
shown in Figure 2. In this experiment, we used a 90 mJ laser energy, and the spot size was 75 µm.
Figure 2c shows the surface morphology of a single point measurement, which was visualized using
scanning electron microscopy. The laser ablation area comprises an ellipse with a longer diameter
of approximately 120 µm, which may be generated by the leaf tissue structure. The ablation depth
was approximately 12 µm, which was measured using atomic force microscopy and scanning electron
microscopy. Figure 2b shows a photograph collected after scanning 20 × 20 points and 4 planes. In this
measurement, the distance between each spot was 200 µm to avoid overlap and an interrelationship.
Thus, the scanning area for a single plane was 4 × 4 mm. When scanning for the first plane was
completed, the 3-dimensional platform moved 12 µm up to scan the next plane. Figure 2b shows
that the laser ablation produced only a small amount of damage to the maize leaves, which may not
influence its growth.
Figure 2d shows a plasma emission spectrum that corresponds to Figure 2b. The elemental
concentrations can be calculated based on the atomic and ionic spectral characteristics [28]. Moreover,
certain spectral characteristics belong to the molecular fragments, e.g., CN, which can be used to
directly analyze the concentrations of certain specific molecules [41]. LipsImag can be used to image
the distributions for the presence and concentrations of certain nutritional elements, e.g., Mg, Ca, and
Na, and for toxic elements at trace concentrations (such as P in pesticide). Figure 3 shows the mapping
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results
forNa,
theand
small
leaf measurements,
in which the
distributions
of theFigure
spectral
intensities
and
for holly
toxic elements
at trace concentrations
(such
as P in pesticide).
3 shows
the for
mapping
results
for the
small holly
in which
the distributions
of theintensities
spectral are
Mg, Ca,
and Na
are clear.
Notably,
theseleaf
aremeasurements,
the pre-calibration
results,
and the spectral
forelemental
Mg, Ca, and
Na are clear. Notably,
these are
the pre-calibration
results,before
and thefield
spectral
used intensities
instead for
concentrations.
In practice,
a calibration
is performed
use and
intensities
are
used
instead
for
elemental
concentrations.
In
practice,
a
calibration
is
performed
beforeusing
is based on samples with known concentrations that are used to construct a quantitative model
field use and is based on samples with known concentrations that are used to construct a quantitative
spectral
characteristics and elemental concentrations. Next, the calibration model was installed in the
model using spectral characteristics and elemental concentrations. Next, the calibration model was
LipsImag software, through which the elemental concentration distributions can be imaged in real
installed in the LipsImag software, through which the elemental concentration distributions can be
time.imaged
Notably,
LipsImag operates at a much higher spatial resolution, e.g., 20 µm, but the sensitivity
in real time. Notably, LipsImag operates at a much higher spatial resolution, e.g., 20 μm,
and detection
limit areand
lower
due tolimit
the laser
ablation
mass
Wemass
will use
LipsImag
with
leaves
but the sensitivity
detection
are lower
due to
the decrease.
laser ablation
decrease.
We will
use
to detect
pesticide
residues
that are
considered
thus,
weelements;
used a 200
µm
LipsImag
with leaves
to detect
pesticide
residuestrace
that elements;
are considered
trace
thus,
weresolution
used
throughout
the
analysis.throughout the analysis.
a 200 μm
resolution
Figure
3. The
mapping
results
holly(Ilex
(Ilexchinensis
chinensis Sims)
onon
thethe
spectral
intensities
of of
Figure
3. The
mapping
results
ofofholly
Sims)leaf
leafbased
based
spectral
intensities
specific
elements
using
LipsImag.
Thevisible
visible image
image of
thethe
mapping
experiment;
(b) and
specific
elements
using
LipsImag.
(a)(a)The
ofthe
theleaf
leafforfor
mapping
experiment;
(b) and
(c) shows the distributions of the spectral intensities of Mg and K, respectively; (d,e) illustrate the
(c) shows
the distributions of the spectral intensities of Mg and K, respectively; (d,e) illustrate the
spectral characteristics of Mg and K that were used for mapping.
spectral characteristics of Mg and K that were used for mapping.
3.2. Chlorpyrifos Residue Spectral Characteristics in Plants and Quantitative Analyses Thereof
3.2. Chlorpyrifos Residue Spectral Characteristics in Plants and Quantitative Analyses Thereof
As discussed previously, measuring pesticide distributions in plant tissue is important for
As
discussed
previously,research.
measuring
in plant
is can
important
for plant
plant
and environmental
Thepesticide
previous distributions
mapping method,
i.e., tissue
MALDI,
only detect
pesticides on theresearch.
leaf surface,
inside ofmapping
a leaf [26].method,
More importantly,
these
methods
are difficult
and environmental
Thenot
previous
i.e., MALDI,
can
only detect
pesticides
to leaf
use surface,
for mapping
in-fieldofand
in vivo
to importantly,
their complex these
structure.
Most are
pesticides
contain
on the
not inside
a leaf
[26].due
More
methods
difficult
to use for
specific
elements
(e.g.,
P
and
S),
which
thus
provides
an
opportunity
for
mapping
the
elements’
mapping in-field and in vivo due to their complex structure. Most pesticides contain specific elements
concentrations in plant tissues using LipsImag [30,42]. In this study, we used chlorpyrifos, which is
(e.g., P and S), which thus provides an opportunity for mapping the elements’ concentrations in plant
a common pesticide in China, as an example and attempted to collect 3-dimentional images of its
tissues using LipsImag [30,42]. In this study, we used chlorpyrifos, which is a common pesticide in
distribution in plant leaves.
China, as Chlorpyrifos
an example (C
and
attempted to collect 3-dimentional images of its distribution in plant leaves.
9H11Cl3NO3PS) contains several elements, including P, S, H, Cl, O, N, and C. H,
Chlorpyrifos
(C
H
Cl
contains
several
including
P,the
S, H,
Cl, O,ofN,
and C.
9 11in air
3 NO
3 PS)
C, O, and N also exist
and
plant
tissue. Thus,
weelements,
attempted to
determine
presence
and
H, C, O, and N also exist in air and plant tissue. Thus, we attempted to determine the presence
of and calculate the concentrations of chlorpyrifos using the spectral characteristics of P, S, and
Cl. Figure 4 shows the plasma emission spectra from LipsImag for a leaf with 20 µg/cm2 and
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calculate the concentrations of chlorpyrifos using the spectral characteristics of P, S, and Cl. Figure 4
200 ng/cm2 chlorpyrifos residue and a clean leaf. The spectral characteristics
of P at 213.62 nm,
shows the plasma emission spectra from LipsImag for a leaf with 20 μg/cm2 and 200 ng/cm2
214.91
nm,
253.56
nm,
and
255.33
nm
are
shown
in
Figure
4a,b,
and
the
spectral
characteristics of
chlorpyrifos residue and a clean leaf. The spectral characteristics of P at 213.62 nm, 214.91 nm,
Cl at 253.56
827.59nm,
nm and
are shown
in Figure
4c. The
spectral
characteristics
for Pcharacteristics
at the four wavelengths
255.33 nm
are shown
in Figures
4a,b,
and the spectral
of Cl at
were 827.59
clear in
the
leaf
samples
with
pesticide
but
were
absent
in
the
clean
leaf,
and the intensities
nm are shown in Figure 4c. The spectral characteristics for P at the four
wavelengths
were
increased
higher
concentration
samples. but
However,
the Cl in
spectral
line leaf,
was much
weaker
such that
clear with
in the
leaf samples
with pesticide
were absent
the clean
and the
intensities
2
with higher
concentration
samples.
However,
the Cl spectral
line was to
much
weaker
it wasincreased
only observed
in samples
with 20
µg/cm
chlorpyrifos.
We attempted
discern
thesuch
spectral
that it wasofonly
in samples
20 μg/cm2 chlorpyrifos.
attempted
to discern
the
characteristics
the observed
S atom and
ion, butwith
the characteristics
were notWe
found.
Spectral
characteristics
spectral
characteristics
of
the
S
atom
and
ion,
but
the
characteristics
were
not
found.
Spectral
of P and S in the ultraviolet band are much stronger than their visible band characteristics [43–45].
characteristics of P and S in the ultraviolet band are much stronger than their visible band
However,
an ultraviolet spectrometer requires a vacuum environment; thus, it is difficult to use in-field.
characteristics [43–45]. However, an ultraviolet spectrometer requires a vacuum environment; thus, it
Furthermore, to measure the pesticide more precisely, we constructed a multi-variable regression
is difficult to use in-field. Furthermore, to measure the pesticide more precisely, we constructed a
modelmulti-variable
using samples
with a known pesticide concentration and the spectral characteristics of P and Cl
regression model using samples with a known pesticide concentration and the
(Figure
4d).
spectral characteristics of P and Cl (Figure 4d).
Figure 4. The laser-induced plasma spectral characteristics of chlorpyrifos in a leaf. (a) The spectral
Figure 4. The laser-induced plasma spectral characteristics of chlorpyrifos in a leaf. (a) The spectral
characteristics of the element P at 213.62 nm and 214.91 nm; (b) The spectral characteristics of the
characteristics of the element P at 213.62 nm and 214.91 nm; (b) The spectral characteristics of the
element P at 253.56 nm and 255.33 nm; (c) The spectral characteristics of the element Cl at 827.59 nm.
element
P at 253.56 nm and 255.33 nm; (c) The spectral characteristics of the element Cl at 827.59 nm;
(d) The multi-variable linear regression model for chlorpyrifos measurement based on the spectral
(d) The
multi-variable
linear
characteristics
of P and
Cl. regression model for chlorpyrifos measurement based on the spectral
characteristics of P and Cl.
3.3. Two-Dimensional Mapping of Chlorpyrifos on a Leaf Surface
3.3. Two-Dimensional
Mapping
of Chlorpyrifos
ondistribution
a Leaf Surface
We used LipsImag
to image
the pesticide
on a leaf surface after the calibration model
wasused
constructed
andto
installed.
Figure
5a shows
the mapping
for a random
maize
leaf after model
a
We
LipsImag
image the
pesticide
distribution
on results
a leaf surface
after the
calibration
spray application, which included 100 mL chlorpyrifos EC (40.7%) diluted with 50 L water and
was constructed and installed.
Figure 5a shows the mapping results for a random maize leaf after
spayed in a 1 mu (666.7 m2) maize field. This dose is common and is a concentration that prevents
a spray application, which included 100 mL chlorpyrifos EC (40.7%) diluted with 50 L water and
aphids and leaf mites in2China. In this experiment, we stretched the leaves onto the platform 20 min
spayed
inthe
a 1spray
mu (666.7
m ) maize
field. This dose
common
and
a ×concentration
after
and collected
the measurements.
Theisscanning
area
wasis20
20 points with that
a 200prevents
μm
aphids
and
leaf
mites
in
China.
In
this
experiment,
we
stretched
the
leaves
onto
the
platform
20 min
distance between each point. The distributions of certain chlorpyrifos droplets are clear in Figure 5a,
after and
the spray
and
collected
the
measurements.
The
scanning
area
was
20
×
20
points
with
a
200
certain vertical bands may have been caused by the droplets falling under gravity. Figure 5b µm
distance
each point.
Thebut
distributions
of certain
droplets
are clear
Figure 5a,
also between
includes mapping
results
with a lower
pesticidechlorpyrifos
dose at 10 mL
chlorpyrifos
EC in
(40.7%),
and certain vertical bands may have been caused by the droplets falling under gravity. Figure 5b also
includes mapping results but with a lower pesticide dose at 10 mL chlorpyrifos EC (40.7%), which
was also diluted with 50 L water. Therefore, the pesticide residue in Figure 5b is at much lower levels
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which was also diluted with 50 L water. Therefore, the pesticide residue in Figure 5b is at much lower
levelsinthan
in 5a.
Figure
5a. We
also
used LipsImag
the processes
of chlorpyrifos
spread
and
than
Figure
We also
used
LipsImag
to study to
thestudy
processes
of chlorpyrifos
spread and
transport
transport
on
a
leaf
surface
through
mapping
a
leaf
10
h
after
spraying
(100
mL
chlorpyrifos
EC),
as
on a leaf surface through mapping a leaf 10 h after spraying (100 mL chlorpyrifos EC), as shown in
shown 5c.
in Figure
5c.the
Clearly,
theconcentrations
droplet concentrations
are but
weaker,
but the
area is greater.
Furthermore,
Figure
Clearly,
droplet
are weaker,
the area
is greater.
Furthermore,
certain
certain
horizontal
bands
appear,
which
may
be
caused
by
water
transport
in
the
leaf
vein.
horizontal bands appear, which may be caused by water transport in the leaf vein.
Figure 5.
5. The
The 2D
2D mapping
mapping results
results of
of chlorpyrifos
chlorpyrifos on
on aa leaf
leaf surface
surface using
using LipsImag.
LipsImag. (a)
(a) The
The mapping
mapping
Figure
result
of
pesticide
on
a
leaf
that
used
100
mL
chlorpyrifos
EC
(40.7%)
diluted
by
50
L
water
and was
was
result of pesticide on a leaf that used 100 mL chlorpyrifos EC (40.7%) diluted by 50 L water and
2) maize field; (b) The mapping result of a lower concentration
2
subsequently
sprayed
in
a
1
mu
(666.7
m
subsequently sprayed in a 1 mu (666.7 m ) maize field; (b) The mapping result of a lower concentration
of pesticide
pesticide that
that used
used 10
10 mL
mL chlorpyrifos;
chlorpyrifos; (c)
(c) The
The mapping
mapping result
result of
of the
the leaf
leaf 10
10 hh after
after spray
spray application
application
of
that
used
100
mL
chlorpyrifos
EC.
The
left
image
of
(a–c)
shows
the
visible
image
observed
by
that used 100 mL chlorpyrifos EC. The left image of (a–c) shows the visible image observed by
microscopy,
while
the
right
image
is
the
scanning
results
that
show
the
distribution
of
the
pesticide.
microscopy, while the right image is the scanning results that show the distribution of the pesticide.
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3.4. Three-Dimensional Mapping of Chlorpyrifos in Leaves
LipsImag
3-dimensional elemental
LipsImag can provide 3-dimensional
elemental mapping
mapping for
for laser
laser ablation
ablation depth
depth [27,35–37,45].
[27,35–37,45].
As discussed above,
above, the
the laser
laser ablation
ablation depth
depth of
of the
the maize
maize leaf
leaf is
is approximately
approximately 12
12 μm
µm with
with a 90 mJ
laser and a 75 µm
μm spot
spot size.
size. The experiment was performed 10 h after spraying (100 mL chlorpyrifos
EC). We
Weimaged
imagedthe
thechlorpyrifos
chlorpyrifos
distribution
a leaf
through
scanning
one plane
after another
distribution
in ainleaf
through
scanning
one plane
after another
with a
with
12 μm thickness
in each
thicker
near the
middle
of the
leaftowas
used
to avoid
12
µma thickness
in each plane.
A plane.
thickerApart
near part
the middle
of the
leaf was
used
avoid
penetration.
penetration.
Figure
6c shows
mapping
results for
pesticide
that were
than
Figuremay
5a,
Figure 6c shows
mapping
results
for pesticide
residues
thatresidues
were thicker
thanthicker
in Figure
5a,inwhich
which
dueenrichment
to the easyof
enrichment
of in
thethe
droplets
ofpesticide
the leaf. concentration
The pesticide
be due may
to thebeeasy
the droplets
middlein
of the
the middle
leaf. The
concentration
clearly
with
depth,
and almost
pesticide
wasthe
detected
fromTothe
fifth
clearly deceased
with deceased
depth, and
almost
no pesticide
wasnodetected
from
fifth plane.
deeply
plane.
To deeply
understand
the processes
of pesticide
permeation
in plants, we
used
a horizontal
understand
the processes
of pesticide
permeation
in plants,
we used a horizontal
band
in Figure
6b and
band
Figure 6b andimage
drew to
a show
cross-sectional
image
to show the
distribution
in aClearly,
lateral
drew aincross-sectional
the pesticide
distribution
in apesticide
lateral plane
(Figure 6c).
plane
(Figure
6c).
Clearly,
the
chlorpyrifos
gradually
decreased
and
disappeared
at
a
50
μm
depth.
It
the chlorpyrifos gradually decreased and disappeared at a 50 µm depth. It is known that the laser
is
knownwill
thatchange
the laser
ablation properties
will changeofthe
propertiesthe
of measurements
the leaf and influence
the
ablation
the physical
the physical
leaf and influence
among two
measurements
among
two layers.
However,
the influence
was not toowhen
much we
in our
experiment
layers. However,
the influence
was
not too much
in our experiment
used
12 µm as awhen
step
we
12 μmTo
asdemonstrate
a step size in the
depth.
To demonstrate
the above Figure
issue, we
6b in but
the
sizeused
in depth.
above
issue, we reproduced
6b reproduced
in the sameFigure
conditions
samean
conditions
with an opposite
direction
the The
bottom
to the top).
distribution
with
oppositebut
measuring
directionmeasuring
(from the bottom
to (from
the top).
distribution
of The
pesticide
shows
of
pesticide
shows
a similar
shape
in thatonly
thatappears
the pesticide
in the first three layers.
a similar
shape
in that
that the
pesticide
in theonly
first appears
three layers.
Figure 6. The 3-dimensional mapping
mapping of
of pesticide
pesticide residues
residues in
in aa maize
maize leaf
leaf 10
10 h after
after spray. (a) is the
visible image
image of
ofthe
thescanning
scanningarea
areaobserved
observedbyby
microscopy;
(b)
is
a
cross-sectional
image
shows
microscopy; (b) is a cross-sectional image
thatthat
shows
the
the pesticide
distribution
in a lateral
(c) the
shows
the 3-dimensional
of the pesticide
by
pesticide
distribution
in a lateral
plane; plane;
(c) shows
3-dimensional
image ofimage
the pesticide
by scanning
scanning
plane
afterwith
another,
with a thickness
of 12
μmplane.
for each plane.
one planeone
after
another,
a thickness
of 12 µm for
each
3.5.
in Soil
Soil Using
Using LipsImag
LipsImag
3.5. Measurement
Measurement of
of the
the Element
Element Concentrations
Concentrations in
To
providean
anability
abilitytotofurther
further
understand
element
transportation
processes
among
soil
To provide
understand
thethe
element
transportation
processes
among
soil and
and plant
tissues,
we also
the LipsImag
system
to detect
element
presence
concentrations
plant
tissues,
we also
usedused
the LipsImag
system
to detect
element
presence
andand
concentrations
in
in
soil.
The
soil
was
sampledand
andsquashed
squashedinto
intoaatablet
tablettotoavoid
avoidsplashing.
splashing. We
We studied
studied the
the spectral
spectral
soil.
The
soil
was
sampled
characteristics
of K,
K, Mg,
Mg,Na,
Na,and
andCa.
Ca.and
andthe
thecalibration
calibrationmodels
modelswere
were
built.
The
concentration
Si
characteristics of
built.
The
concentration
of of
Si in
in
is stable,
soused
we the
used
the spectral
line atnm
288.16
ofinternal
Si as an
internaland
standard
and
soilsoil
is stable,
so we
spectral
line at 288.16
of Si nm
as an
standard
established
established
calibration
models
to
reduce
the
influence
of
laser
jitter
and
soil
moisture.
Figure
7
calibration models to reduce the influence of laser jitter and soil moisture. Figure 7 shows the spectral
shows
the
spectral
characteristics
and
the
calibration
model
of
K
in
soil.
characteristics and the calibration model of K in soil.
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Figure
Figure 7.
7. Determination of K in soil using LipsImag. (a) shows the spectral characteristics of K in soil;
(b)
(b) is
is the
the calibration
calibration model
model for
for K
K concentration
concentration measurement.
measurement.
4. Conclusions
Conclusions
This study offers a new elemental mapping method to botanists besides X-ray microscopy-based
methods, mass
probes.
Because
it can
be moved
to the
mass spectroscopy
spectroscopybased-methods,
based-methods,and
andfluorescent
fluorescent
probes.
Because
it can
be moved
to
field
and and
can measure
livingliving
plantsplants
directly,
the distribution
of nutrients
and harmful
elementselements
in plant
the field
can measure
directly,
the distribution
of nutrients
and harmful
tissues
well asasthe
interactions
betweenbetween
plants and
the environment
can be studied
without
in plantastissues
well
as the interactions
plants
and the environment
can benaturally
studied naturally
withoutcultivation,
sample cultivation,
and pre-treatment.
Theablate
laserthe
will
ablateduring
the sample
during
sample
collection,collection,
and pre-treatment.
The laser will
sample
measurement,
measurement,
butarea
theisablation
areawill
is small
and will the
not plant
influence
the plant
it can be
but
the ablation
small and
not influence
growth;
thus, growth;
it can bethus,
considered
a
considered a micro-destructive
or even non-destructive
measurement
The
above experiments
and
micro-destructive
or even non-destructive
measurement
tool. The tool.
above
experiments
and results
results demonstrate
that LipsImag
can provide
3-dimensional
mapping
for elemental
distributions
in
demonstrate
that LipsImag
can provide
3-dimensional
mapping
for elemental
distributions
in living
livingtissues.
plant tissues.
MALDI-MS
also
for pesticide
imaging
in plant
leaves
withresolution
a spatial
plant
MALDI-MS
was alsowas
used
forused
pesticide
imaging in
plant leaves
with
a spatial
resolution
of300
300µm
μm[26],
× 300
μm [26],
which
is lower
thanMALDI-MS
LipsImag. MALDI-MS
images the
pesticide
of
300 µm ×
which
is lower
than
LipsImag.
images the pesticide
distribution
distribution
based structure
on molecular
structure
while
LIBS is
based on atomic
concentration.
More
based
on molecular
while LIBS
is based
on atomic
concentration.
More importantly,
LipsImag
importantly,
LipsImag
hasstructures
much simpler
opticaltostructures
compared
to can
MALDI-MS
and
can be
has
much simpler
optical
compared
MALDI-MS
and thus
be moved
to thus
the field
to
moved to
field todistribution
measure the
elemental
distribution
of living to
plants.
LIBS isbecause
comparable
to
measure
thethe
elemental
of living
plants.
LIBS is comparable
LA-ICP-MS
both are
LA-ICP-MS
because
both
are based
on laser
A main
restriction
for LA-ICP-MS
applications
based
on laser
ablation.
A main
restriction
for ablation.
LA-ICP-MS
applications
in plant
research is dehydration
in plant
research
dehydration
effects;
related conditions
studies have
experimental
effects;
thus,
relatedisstudies
have focused
on thus,
experimental
that focused
maintain on
sample
moisture
conditions
that
maintain
sample this
moisture
andisstructure
[2,22,23].
However,tothis
problem
is avoided
and
structure
[2,22,23].
However,
problem
avoided by
using LipsImag
directly
measure
living
by using
LipsImag
toconditions.
directly measure
livingLipsImag
plants under
growth
conditions.
Moreover, LipsImag
plants
under
growth
Moreover,
includes
certain
other advantages,
such as a
includes certain
other of
advantages,
such asand
a simultaneous
analysistoof
multiple
elements and low
simultaneous
analysis
multiple elements
low cost, compared
other
methods.
cost, Notably,
compared
to is
other
methods.
this
the first
study where LIBS was applied to in situ and in vivo elemental mapping
Notably,
this
is the first
study
LIBS was
applied
to we
in situ
and
mapping
of plants;
thus,
further
studies
arewhere
necessary.
In this
study,
used
a in
200vivo
µmelemental
spatial resolution,
of plants;
thus, further
studies are
necessary.
In this
weand
used
a 200
μm spatial
resolution,
which
which
is lower
than previously
used
methods,
e.g.,study,
SRXRF
SIMS
[8,9,16].
In fact,
LipsImag
can
is lowerat
than
previously
methods,
e.g.,
SRXRF
and smaller
SIMS [8,9,16].
fact,cells
LipsImag
can operate at
operate
a much
higher used
resolution
(10–50
µm)
to image
objects,Ine.g.,
and microorganisms.
a much
resolution
(10–50
image
smaller objects,
cells and
We
We
used higher
a 200 µm
resolution
due toμm)
the to
trace
concentrations
of thee.g.,
pesticide
in themicroorganisms.
plant leaves, which
useddifficult
a 200 μm
dueatolower
the trace
concentrations
of the pesticide
in the
leaves, which
are
to resolution
detect using
energy
laser and smaller
spot size.
Weplant
will attempt
to useare
a
difficultresolution
to detect in
using
a lower
energy
laser and
smaller
spot size.
will
attempt
to useissue
a higher
higher
future
studies
for mapping
nutrient
elements
in We
plant
tissues.
Another
that
resolution
in future studies
for mapping
elements
in plant
Another
must
must
be considered
is the detection
limitnutrient
of the LIBS
method,
whichtissues.
can reach
ppm issue
levelsthat
for solid
be considered
the detection
limit of [28].
the LIBS
method,
which
can reach buffered
ppm levels
for and
solid
analyses
under isconventional
conditions
Using
a vacuum
environment,
gases
a
analyses under
[28]. Using
a vacuum
buffered
and
multi-pulse
laserconventional
can enhance conditions
the LIBS detection
sensitivity
but environment,
lead to difficulties
with gases
in-field
anda
multi-pulse
laser can enhance
theDuring
LIBS detection
sensitivity butprocess,
lead tothe
difficulties
with in-field
and
in
vivo measurements
[27,28,30].
the LIBS measurement
plasma spectrum
contains
in vivo characteristics
measurementsof[27,28,30].
During
the LIBS
measurement
thethe
plasma
spectral
atom, iron,
and molecule
fragments,
but weprocess,
only used
atomicspectrum
spectral
contains
spectral
characteristics
of atom,
iron, and molecule
fragments,
but weofonly
used the
atomic
line
in this
study. For
certain specific
measurements,
the spectral
characteristics
molecular
fragments
spectral
line in this
study.
the spectral characteristics
of molecular
can
be observed
and
usedFor
to certain
directlyspecific
image measurements,
the molecular distributions
[41,46–48]. Theoretically,
fragments
can be nearly
observed
and used but
to it
directly
image for
theelements
molecular
distributions
LIBS
can measure
all elements,
is a problem
that
exist in air, [41,46–48].
e.g., C, H,
Theoretically,
LIBS can
measure
nearly
all elements,
it isusing
a problem
for elements
that or
exist
in air,
and
O. One potential
way
to avoid
the influence
of airbut
is by
a vacuum
environment
buffered
e.g., C, H, and O. One potential way to avoid the influence of air is by using a vacuum environment or
buffered gases [49]. Other methods may also be used to measure these elements, such as establishing
Sensors 2016, 16, 1764
11 of 13
gases [49]. Other methods may also be used to measure these elements, such as establishing particular
models based on chemometrics, which must be considered under specific conditions and for specific
applications [28,48].
Acknowledgments: This work was supported by the National Natural Science Foundation of China (No. 61134011,
31271614 & 31622040).
Author Contributions: Chunjiang Zhao wrote the main manuscript text, Daming Dong designed the project,
Wengang Zheng and Xiaofan Du performed the experiment, Chunjiang Zhao and Daming Dong performed the
calculations. All authors reviewed the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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