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

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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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