Laser-induced breakdown spectroscopy as a promising tool in the elemental bioimaging of plant tissues

Journal Pre-proof Laser-induced breakdown spectroscopy as a promising tool in the elemental bioimaging of plant tissues Pavlína Modlitbová, Pavel Pořízka, Jozef Kaiser PII:

S0165-9936(19)30586-2

DOI:

https://doi.org/10.1016/j.trac.2019.115729

Reference:

TRAC 115729

To appear in:

Trends in Analytical Chemistry

Received Date: 8 October 2019 Revised Date:

5 November 2019

Accepted Date: 5 November 2019

Please cite this article as: P. Modlitbová, P. Pořízka, J. Kaiser, Laser-induced breakdown spectroscopy as a promising tool in the elemental bioimaging of plant tissues, Trends in Analytical Chemistry, https:// doi.org/10.1016/j.trac.2019.115729. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier B.V. All rights reserved.

Laser-induced breakdown spectroscopy as a promising tool in the elemental bioimaging of plant tissues Pavlína Modlitbová1, Pavel Pořízka1, Jozef Kaiser1* 1

Central European Institute of Technology (CEITEC) Brno University of Technology, Technická 3058/10, 616 00 Brno, Czech Republic

*Corresponding author: Jozef Kaiser [email protected] Central European Institute of Technology (CEITEC) Brno University of Technology Technická 3058/10 616 00 Brno Czech Republic

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ABSTRACT Laser-Induced Breakdown Spectroscopy (LIBS) is an optical analytical technique with a multi-element capability for element bioimaging in plants. During the years of LIBS development, the major application field has been in industry. However, during the last two decades, LIBS became a useful imaging tool in various biologic matrices, e.g. bones, mammals’ organs, and in the plant science. In this work, we present an overview of LIBS achievements in plant bioimaging which started in 2006. The progress in the assessment of spatial element distribution in plants is documented here with respect to the applications in phytotoxicity testing for the following reason: the information on the spatial distribution of elements can reveal a relationship between the exact location of an element and its toxic effect. This review discusses the state of the art of various elements’ bioimaging in plants using LIBS with a spatial resolution at micrometer scale.

KEYWORDS crop plants, elemental imaging, element spatial distribution, laser ablation, heavy metals, macronutrients, micronutrients, model organisms, nanoparticles, plants, phytotoxicity, 2D-mapping

HIGHLIGHTS Means to implement LIBS into the toxicological routine are discussed. Benefits and drawbacks of LIBS are put into context with respect to other analytical techniques. Plant exposure to compounds and nanoparticles follows by plant preparation prior LIBS analysis is investigated in detail. Extending information on the influence of nanoparticles on plant organisms gained from LIBS analysis are showed. Imaging of nanoparticles in plant tissue, a novel trend in laser-induced breakdown spectroscopy is reviewed.

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1. Introduction Plants, as primary producers that convert light and inorganic compounds into organic, are influenced by the quality and chemical composition of soil, water, and atmosphere which they are in contact with. There is a need to acquire comprehensive information about possible effects of elements, compounds, and even micro- and nanomaterials on the growth and development of plant tissues. This knowledge is important for a sustainable agricultural production, ensuring the nutritional quality and safety of products, and the most importantly for assessing negative effects on plant tissues and cell-levels. Plant nutrient research and plant toxicology aim to understand the processes induced in plant tissues with varying contents of selected essential and non-essential chemical compounds. Moreover, the goal of these research fields is to understand how the selected essential and nonessential chemical compounds are acquired, transported, distributed, stored, and used in plant tissues [1–3]. Plant nutrient research and plant toxicology exploit primarily the traditional analytical techniques (such as atomic absorption/emission spectroscopy; AAS/OES, or mass spectrometry; MS) that are used to estimate the chemical composition of the bulk material, after acid digestion [4,5]. Thus, the estimation of chemical composition of the investigated plant together with the bioaccumulation of chemical compound is highly accurate and sensitive. However, detailed information about the distribution of investigated compounds is either lost or it is attributable only to a part of the plant (e.g. root, stem, leaves). Recently, chemical analysis has shifted from the bulk information discussed above to the spatially resolved information obtained by mapping of sample surfaces. The so-called “standard” elemental imaging methods are laser-ablation inductively-coupled plasma mass spectroscopy (LA-ICP-MS) [6], secondary-ion mass spectrometry (SIMS) [7], scanning electron microscopy with energy-dispersive Xray spectroscopy (SEM-EDX) [8], particle-induced X-ray emission (PIXE) [8], and synchrotron-based techniques [8,9] such as X-ray absorption near-edge spectroscopy (XANES), extended X-ray absorption fine structure (EXAFS), and micro-X-ray fluorescence (µXRF). In contrast to these methods, Laser-Induced Breakdown Spectroscopy (LIBS) is presented here as a novel analytical tool for multi-elemental plant bioimaging with a high resolution (micro-scale) on a large-scale sample (whole slide imaging). LIBS represents a vital alternative to its analytical counterparts and delivers complementary performance (large scale mapping). LIBS, an analytical method based on the optical emission, has developed in recent years to become a fully unassisted technique. It has gained popularity in modern analytical chemistry because of its short analysis time, multi-elemental capability (simultaneous detection of broad range of elements, including light elements and halogens), little if any sample preparation, and remote and in situ sensing. The capability to assess coarse spatial distribution of elements became the biggest benefit of LIBS [10]. A brief introduction to LIBS principles, methodology, and instrumentation is provided in the following section of this review, whereas a comprehensive description of the technique can be found in earlier reviews [10–12]. Because of the undeniable advantages of LIBS, great progress could be achieved in the analysis of various biological materials, as it was recently summarized by Busser et al. (2018) and Jolivet et al. (2019) [13,14]. The elemental spatially resolved LIBS analysis was already published in various biological and medical studies, especially by Vincent Motto-Ros’s research group. The spatial distribution of elements in mouse kidneys [15-20], mouse tumours [21,22], healthy human skin or human skin with tumours [23], and human lung tumour [24] was already described. However, most importantly, 15 original research papers were published between 2006 and 2019 that reported using

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LIBS to map various elements, materials, and compounds in several plant species (terrestrial and aquatic) and different plant parts (shoots, leaves, and roots) [25–39]. Early stages of LIBS use for the analysis of plant material focused on the assessment of macro- and micronutrient content, as these nutrients are needed in agriculture to ensure a proper plant growth [40–43]. LIBS was supposed to be further used for in situ analyses in agriculture, however, instead of analyzing the nutrients content, the focus shifted elsewhere. Due to a rising production of various new materials and compounds in the last decades it was necessary to evaluate possible negative impact of these materials on the environment. A demand has also arisen for a novel analytical tool capable of determining the precise spatial distribution of elements in model organisms. The first studies using LIBS to assess the distribution of various contaminants in plants already correlated it to the plants’ physiological pathways. For example, three studies successfully mapped the distribution of various nanoparticles (NPs) in plant organs, namely CdTe NPs, Ag NPs, and photon-upconversion NPs, and correlated their uptake and toxic effects to the location determined by LIBS [34,35,37]. However, these papers also emphasized a need for a reference/complementary method that would help to obtain more detailed data. This review describes in detail the novelty of using LIBS as an element bioimaging technique in the analysis of plant samples and highlights the value of the assessment of spatial element distribution in phytotoxicity testing. The review is divided into three sections. The first section briefly discusses basic information about the use of LIBS in the analysis of plant samples and describes typical LIBS set-ups and arrangements, data evaluation, and sample preparation. The second and the most important part focuses on the element distribution of macronutrients (K, Ca, Mg, and P) [28–33,36], micronutrients (Fe, Mn, Cu, and B) [27,28,30–32,34], non-essential elements (Pb, Cd, Ag, Si, Li, Y, Yb, and Cr) [25–30,32,34–39], NPs (CdTe NPs, Ag NPs, and photon-upconversion NPs) [34,35,37], and organic compounds (chlorpyrifos) [23,29] in various model plants. The third section compares LIBS to the most commonly used analytical techniques in plant bioimaging and critically discusses the differences among the methods together with their benefits and drawbacks. Finally, the conclusion addresses the challenges, environmental implications, and wider use of LIBS.

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2. The use of laser-induced breakdown spectroscopy in the analysis of plant samples 2.1 Plant exposure Phytotoxicity of various contaminants is commonly assessed with standard toxicity tests (e.g., Organization Economic Co-operation and Development, OECD; International Organization for Standardization, ISO; and United States Environmental Protection Agency, US EPA) [44] or nonstandard tests where the exposure has to be described in detail [45]. Usually, toxic effects to plants or their parts are evaluated at the organism/tissue/cell levels. However, the distribution, bioaccumulation, uptake, and translocation of tested compounds are evaluated at the end of exposure. The influence of selected contaminants can be unraveled more precisely if all results are evaluated as a whole. Furthermore, the information on the spatial distribution of elements can reveal the relationship between the exact location of an element and its toxic effect. Plants can be exposed to test compounds in different ways. Terrestrial plants (e.g., bell pepper, Capsicum anuum; common sunflower, Helianthus annuus; Asian rice, Oryza sativa; radish, Raphanus sativus; broad bean, V. faba) were most commonly subjected to well-defined toxicity tests, such as various solutions/dispersions in hydroponic conditions for exact time periods (from several hours to months). Macroscopic toxicity endpoints were set, and the plants were processed for various analyses at the end of exposure [25–30,34,35,37,38]. Differently, Krajcarová et al. (2013) used a simple exposure of European spruce (Picea abies) needles to CuCl2 solutions with different concentrations (1/3 of a needle was dipped into the solution) to evaluate the spatial distribution of Cu and Ca in cross-sections of terminal stems [31]. A similar experiment has recently been done by Singh et al. (2019) who immersed the leaves of southern yew (Podocarpus macrophyllus) into a LiCl solution for 8–48 h to evaluate the diffusion of Li into leaves [36]. Aquatic plants were also tested, common duckweed (Lemna minor) was exposed for 168 hours to selected xenobiotics, and its fronds were mapped with LIBS to distinguish between the distribution of NPs and ions [35,37]. The two exposures differed a lot, firstly the plants were exposed to CdTe NPs dispersions/Cd2+ solutions (diluted in Steinberg medium, volume 150 mL) [37] in the toxicity according to the OECD Test No. 221: Lemna sp. Growth Inhibition Test [46] and later, the miniaturized duckweed test [37] using only 3 mL of test media (Y3+ and Yb3+ solutions/ photon-upconversion NPs dispersion in Milli-Q water) was constructed in culture plates [37]. Test compounds were also directly applied onto plant surface. A solution of chlorpyrifos (C9H11Cl3NO3PS) was sprayed onto maize leaves in the field, and its diffusion through leaves was assessed after 10 h by mapping several layers of one leaf. The publication featured the first attempt to create a 3D model of plant leaf from several 2D maps [33]. In another study, 20-µL drops of chlorpyrifos or Cd(NO3)2 were applied directly onto the surface of lettuce (Lactuca sativa) and chives (Allium schoenoprasum) and were analyzed using LIBS after air-drying [39]. However, these studies do not provide the information on the real uptake or distribution of tested compounds in plants, only on their possible detection. On the contrary, Guerra et al. (2015) analyzed leaves of sugar cane (Saccharum spp.) collected in the field and showed that they contained naturally occurring essential elements (P, Ca, Mg, Fe, Mn, and B) and the background of non-essential element (Si) [32]. However, similarly to other agricultural studies, this paper has no toxicological message [42]. 5

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2.2 Sample preparation One of the most emphasized advantages of LIBS is the fact that it requires no or minimal sample preparation. Unfortunately, this is not true for biological samples. Only a few easy procedures for biological sample preparation have been published so far. The most common, fast, and economical procedure is drying of molded samples followed by their embedding in epoxy or double-sided adhesive tape to glass slides [25,32,35,37–39]. Plant leaves can be quickly molded, freezed, and then analyzed [27,30]. Unfortunately, 2D maps obtained in those experiments could not discern the elemental distribution inside the plant tissues, the particle absorption on the plant surface, or the exact uptake pathways. In order to acquire values of these features, the measurements must be complemented with results from other techniques. For example, the distribution of rare earth element NPs in R. sativus was observed indirectly via the signal of Y and Yb that they contain. In fact, only the information on the distribution of Y and Yb was acquired with LIBS, and their origin from photon-upconversion NPs had to be confirmed with photon up-conversion scans [37]. Contrary to mammalian tissues, plants were rarely cut into cross-sections for LIBS analysis [31,34]. This was due to time-consuming and unoptimized procedures, as well as a somewhat low spatial resolution of measured maps (from 750 µm down to 50 µm step sizes). The first attempt to prepare cross-sections was by cutting spruce into 1‒2 mm thick sections, followed by their fixing with glycerol gel onto glass slides [31]. The best spatial resolution published so far (50 µm) was achieved by preparing cryo cross-sections (20 µm, 40 µm, and 100 µm) as well as classic paraffin cross-sections (15 µm, 20 µm, 40 µm, 80 µm, and 100 µm) of V. faba roots. In that study, the thickness of 40 µm was estimated as optimal for elemental mapping. Simultaneously, cryo cross-sectioning was selected as the best method for root preparation for its speed, simplicity, and preservation of key parts of the sample structure [34]. Spatial distribution of P, Cl, Mg, K was assessed directly in situ only once [33], namely with a removable LIBS system (LipsImag) in leaves of common corn (Zea mays) and purple holly (Ilex chinensis). Almost no sample preparation was needed – fresh leaves were simply fixed with adhesive tape onto the platform [33]. The sample preparation is dependent on the expected result, if we are interested in the analysis of the whole organisms, the dried molded plants fixed in proper medium is a satisfactory preparation method. Also, the spatial resolution of LIBS analysis will be then chosen based on the sample size. If detailed data for specific plant parts are demanded, the cross-section preparation seems to be necessary. However, an optimization of detail preparation procedure is necessary and should be dealt with as the first step of future research studies. Also, with the expected improvement of lateral resolution, LIBS analysis on cellular level will need a new robust and complex approach in the preparation as well. 2.3 Instrument set-up LIBS instrumentation is relatively simple and thus robust. Detailed specifications of the LIBS instrumental components have been discussed elsewhere [10] and only a brief summary is provided here. Technically, a typical LIBS system can be divided into two parts, i) ablation and ii) collection. The lateral resolution of maps in x and y directions is defined by step size (i.e., the distance between two 7

consecutive laser pulses) and is limited by the size of the ablation crater created after the laser pulse ablation. A moving sample holder with a high accuracy and small steps are necessary accessories for mapping. First, the ablation part of a LIBS system relates to the laser itself and its properties. LIBS is a laserablation based technique and is thus significantly influenced by the processes beyond laser-matter interaction, collectively inducing the matrix effect. Material ablation (sampling) is achieved by a laser pulse which is focused on a narrow spot, creating laser-induced plasma. Properties of a laser pulse (wavelength, duration, lateral intensity profile, spot diameter) influence the analytical performance of a LIBS analysis. Their influence on ablation and quality of laser-induced plasma is out of the scope of this manuscript, however, they are reviewed in detail in Hahn and Omenetto (2010) [11]. The most commonly used laser in plant analysis is the nanosecond laser (Nd:YAG) with various wavelengths (1064 nm, 532 nm, and 266 nm) and pulse energies (5‒160 mJ) [26–30,32,33,36,38,39]. The femtosecond laser (Ti:sapphire; 795 nm) with an energy of 0.1 mJ per pulse has been used only once so far [25]. Characteristic plasma emission is collected and transmitted through an optical fiber to the entrance slit of the spectrometer, and spectrally resolved light is recorded with a camera and saved for further analysis. The most commonly used spectrometers are in Czerny-Turner or echelle arrangements equipped with CCD or ICCD detectors. The choice of a suitable spectrometer for a particular analysis depends on various factors: the desired sensitivity to be achieved, operation speed, need of portability, need of multi-elemental detection, and also economic aspects have to be considered. Very often, plants were analyzed with LIBS in the nanosecond single-pulse adjustment (SP-LIBS), i.e., by using one laser pulse to obtain an emission spectrum from a single spot. This setting can be used in laboratory conditions as well as in situ [33,37]. The analytical sensitivity was greatly advanced by (i) the improvement of set-up by adding the second laser in the double-pulse arrangement (DP-LIBS) [31,34,35] or, very recently, by (ii) the deposition of NPs (Ag NPs) onto sample surface, which is known as NP-enhanced LIBS (NELIBS) [39]. By using these approaches, limits of detection (LODs) could be improved up to two orders of magnitude. Due to the momentum of recent development, better values of some LIBS properties have been achieved: the repetition rate of LIBS analysis (up to a laser pulse frequency of 100 Hz), acceptable spatial resolution (down to a 50 µm step), and short analysis time of a single analysis (approx. 3 hours to obtain a megapixel image). Recent state-of-the-art instrumentation and methodology enabled the qualitative elemental imaging of sample surfaces with a high resolution (down to the micro-scale) on a large scale (up to the whole-slide image) and the megapixel imaging of the element distribution [13,14]. Also, a possibility to create 3D models of the elemental composition of biological samples was recognized as highly beneficial to the analysis of animal tissues [16]. From the financial point of view, the affordable purchase price of a LIBS set-up, low operating costs, and a relatively simple instrumentation that can be easily built, serviced, or modified in a laboratory are also considered as great advantages. From our point of view, LIBS is a good option for analysis of unknown samples as the so-called "firstchoice method". LIBS provides a timely analysis of the element distribution with an acceptable spatial resolution. In the second step, it can be complemented with other analytical techniques

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providing higher sensitivity or higher spatial resolution with longer turn-around time or on a lower scale than other techniques. 2.4 Data processing Processing obtained spectra is a rather straightforward task because performing only a qualitative analysis within the plant is sufficient in majority of cases. The intensity of analytical line is simply estimated, usually as the peak maximum or the area under the curve fitted to the spectral line, and depicted in a 2D map showing the distribution of an analyte. Showing results of an analysis in a 2D map is common in case of LIBS analysis. On the contrary, a 3D map from LIBS depth profiling was provided only once on low-scale area [33]. There are further means for LIBS data preprocessing, such as the background subtraction, smoothing and standardization. However, there is no robust algorithm applicable to any experimental data and the methodology differs from case to case. We recommend to study the reviews of Hahn and Omenetto (2010, 2012) to explore various possibilities in data processing and to understand nuances in the results that they will lead to [10,11]. It is important to note that data processing algorithm should be efficient and simple due to the increasing number of spectra to be analyzed (reaching from hundreds of thousands to millions of spectra for recent state-of-the-art LIBS systems). 2.5 Quantitation of elemental images Quantitative analysis is of the paramount interest to the spectroscopic community as well as the discussed toxicological application. However, providing an accurate quantification of analyte content in the spot-by-spot LIBS experiment (2D map) is challenging. The reason is the vast heterogeneity of the sample matrix and the lack of any matrix-matched standards. Recently, data processing has been combined with chemometrics in order to overcome the matrix effect and to improve the accuracy of quantitative model [47]. Peng et al. (2019) employed partial least squares regression (PLSR) and support vector machines (SVM) algorithms for prediction of Cr in rice leaves, reaching root mean square error of prediction of 13.4 mg/kg. They used ICP-MS after acid digestion to achieve reference Cr values [38]. This approach is, however, doubtful when the bulk content is correlated with a heterogeneously distributed analyte. Thus, any quantitative models are still under investigation and far from being applicable routinely to LIBS analysis.

3. Applications of element bioimaging in plants Studies concerning the spatial element distribution of various compounds and materials assessed with LIBS are listed in Table 1. Basic information on the analyzed plant species, detected elements, contamination sources, LIBS instrumentation and experimental conditions, spatial resolution, and sample preparation are also briefly specified. Figure 1 illustrates the recent progress in plant bioimaging, clearly visible by comparing a typical 2D LIBS map from a paper published in 2009 to another coming from 2019.

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Tab 1: The summarized research papers dealing with element imaging in plants. Year of publication

Spatial resolution (μm)

Sample preparation

Reference or complementary method

SP fs-LIBS; Ti:sapphire (795 nm), 100 µJ per pulse

100

dried leaves

X-ray radiography

SP LIBS; Nd:YAG (532 nm), 10 mJ per pulse

500

molded dried leaves

/

1

Pb (405.76 nm)/Pb(CH3COO)2

[26]

Helianthus annuus part of leaves

Ag (328.07 nm)/AgNO3 Cu (324.75 nm)/EDTA

SP LIBS; Nd:YAG (532 nm), 10 mJ per pulse

500

molded freezed leaves

LA-ICP-MS

[27]

Helianthus annuus part of leaves

Pb (283.31 nm)/Pb-EDTA Mg (277.98 nm) Cu (324.75 nm)

SP LIBS; Nd:YAG (532 nm), 10 mJ per pulse

200

dried and partially dried leaves

LA-ICP-MS

[28]

Zea mays, Helianthus annuus, Lactuca sativa part of leaves

Pb (283.31 nm)/Pb-EDTA Mg (277.98 nm)

SP LIBS; Nd:YAG (532 nm), 10 mJ per pulse

LA-ICP-MS

[29]

2011

Capsicum annuum part of leaves

Pb (405.78 nm)/Pb(NO3)2 K (404.41 nm, 404.72 nm) Mn (403.07, 403.31 nm)

SP LIBS; Nd:YAG (532 nm), 10 mJ per pulse

500

molded freezed and dried leaves

LA-ICP-MS

[30]

2013

Picea abies needles

Cu (324.75 nm)/CuCl2 Ca (317.93 nm)

DP LIBS; Nd:YAG (266 nm), 10 mJ per pulse, Nd:YAG (1064 nm), 100 mJ per pulse

150

cross sections of needles, 1–2 mm in thickness

ICP-MS, fluorescence microscopy

[31]

2015

Saccharum spp. leaves

P (213.62 nm); Ca (315.89 nm); Mg (277.98 nm); Fe (259.94 nm); Mn (259.37 nm); B (249.77 nm); Si (212.41 nm)

SP LIBS; -2 Nd:YAG (1064 nm), 50 J·cm

750

fragments from dried middle-third leaves

EDXRF

[32]

Zea mays Ilex chinensis part of leaves, leaves

P (213.62 nm, 214.91 nm, 253.56 nm, 255.33 nm), Cl (827.59 nm)/ C9H11Cl3NO3PS Mg (279.75 nm, 280.27 nm) K (766.79 nm, 769.89 nm)

200

Zea mays: stretched fresh plants fixed by adhesive tape onto a platform Ilex chinensis: cut fresh leaves fixed to a platform

/

[33]

Plant species Part of plant

Element (emission lines)/source

Instrumentation

Helianthus annuus leaves

Pb (405.8 nm)/Pb(C2H3O2)2 Cd (326.1 nm)/CdCl2

2007

Helianthus annuus part of leaves

2008

2009

2006

2009

2016

in situ LIBS; Nd:YAG (1064 nm), 90 mJ per pulse

500

dried and partially dried leaves

Ref.

[25]

10

2017

Vicia faba root

Cu (324.75 nm)/CuSO4 Ag (328.07 nm)/AgNO3, Ag NPs

DP LIBS; Nd:YAG (266 nm), 5 mJ per pulse, Nd:YAG (1064 nm), 100 mJ per pulse

50

cryo-cross sections of roots, 40 µm in thickness

ICP-OES

[34]

2018

Lemna minor leaves

Cd (508.58 nm)/CdCl2, CdTe-MPA NPs, CdTe-GSH NPs

DP LIBS; Nd:YAG (266 nm), 10 mJ per pulse, Nd:YAG (1064 nm), 100 mJ per pulse

200

molded dried leaves glued with epoxide

ICP-OES, TEM

[35]

2018

Podocarpus macorophyllus leaves

Li (670.76 nm)/LiCl Ca (612.2 nm)

SP LIBS; Nd:YAG (266 nm), 15 mJ per pulse

256

fresh leaves

/

[36]

2019

Lemna minor Raphanus sativus leaves, whole plant

Y (Y 437.49 nm), Yb (398.79 nm)/YCl3 3+ 3+ + YbCl3, NaYF4:Yb , Er -SiO2-COOH NPs

SP LIBS; Nd:YAG (532 nm), 10 mJ per pulse (L. minor), 20 mJ per pulse (R. sativus)

100

molded dried leaves glued with epoxide

ICP-OES, photon upconversion microscanner

[37]

Oryza sativa leaves

Cr (359.35 nm, 425.43 nm)/ K2Cr2O7

DP LIBS; Nd:YAG (532 nm), 60 mJ per pulse, Nd:YAG (1064 nm), 50 mJ per pulse

n.a.

dried leaves

ICP-MS

Lactuca sativa part of leaves and stem Allium schoenoprasum part of leaves

P (213.62 nm, 214.91 nm, 253.56 nm, 255.33 nm), Cl (827.59 nm)/ C9H11Cl3NO3PS Cd (214.4 nm)/Cd(NO3)2

NELIBS; Nd:YAG (1064 nm), 160 mJ per pulse, AgNPs 80 nm

dried leaves, fixed to a glass slide with double-sided adhesive tape

/

2019

2019

2

300

[38]

[39]

1

No reference/complementary method used. Data not provided.

2

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Fig. 1: (a) Spatial distribution of Pb and Mg in plant leaves: A – common corn (Zea mays), B – common sunflower (Helianthus annuus). (b) Spatial distribution of Y and Yb in a whole plant: Raphanus sativus. (a) Adapted from reference [29] published as an open-access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license. (b) Adapted from reference [37].

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3.1 Essential elements Essential elements relevant to plant analysis are macronutrients (C, H, O, N, P, K, Ca, Mg, and S) and micronutrients (Fe, Cu, Mn, Zn, B, Mo, Cl, and Ni). They are required for a proper plant growth and they might decrease crop yields if not present in appropriate mass fractions in different plant tissues. A spatial distribution of several macronutrients (K, Ca, Mg, and P) and micronutrients (Fe, Mn, Cu, and B) was determined using LIBS for various plant species in their leaves (Z. mays, H. annuus, L. sativa, C. annuum, I. chinensis, P. macrophyllus, Saccharum spp.) [27–30,32,33,36], roots (V. faba) [34], and needles (P. abies) [31]. Except for Cu, all analyzed elements were present in their naturally occurring amounts. In the case of Cu, the plants were artificially spiked with Cu2+ ions. Root cross-sections of V. faba were mapped after a 7-day exposure to 10 µM CuSO4 [34]. In P. abies, 15-cm-long needles were submerged into CuCl2 solutions (1–50 mM Cu) for 2–24 hours and then their thick cross-sections (1–2 mm thickness) were analyzed [31]. The Cu uptake pathways, translocation, and toxicity were then discussed. In V. faba root cortex, the Cu distribution was homogeneous, and the effects of Cu ions were assessed by comparing the roots of control and exposed plants. A shorter root length, different color, and the absence of lateral roots pointed to a detectable toxic effect of the test solution at low concentration of Cu (10 µM) [34]. In P. abies, Cu2+ ions passed from the pith to other tissues, and the highest amount of Cu was detected in locations corresponding to secondary xylem and phloem in the cross-section. Also, a not negligible amount of Cu2+ ions was probably transported through xylem/phloem radial parenchyma to the ground tissues such as the cortex and secondary dermal tissues. The Cu content was the highest near to the solution surface and also in plants exposed to the highest tested concentration (20 mM Cu) for the longest time (24 h) [31]. Other studies concerning the spatial distribution of essential elements detected the macro- or micronutrients only as a part of complementary analysis. The only exception is the simultaneous determination of P, K, Ca, Mg, Fe, Cu, Mn, Zn, B and Si in sugar cane leaves proved in an experiment done by Guerra et al. (2015) who investigated the nutritional status of these plants in the field. However, the main message of their work was the cross-validation between LIBS and EDXRF (energy dispersive X-ray fluorescence spectrometry) data for selected elements [32]. 3.2 Non-essential elements Spatial distribution of non-essential elements (Pb, Cd, Ag, Cr, Li, Y, Yb, and Si) was analyzed after the plants had been exposed to solutions of Pb(C2H3O2)2, Pb(NO3)2, Pb-EDTA, CdCl2, Cd(NO3)2, AgNO3, K2Cr2O7, YCl3, YbCl3, or LiCl in various phytotoxicity tests. Of the naturally occurring elements, only Si was analyzed in sugar cane collected in the field without any target contamination as discussed in the previous section [32]. In the first instance, the distribution of Pb from three different ionic sources (Pb(C2H3O2)2, Pb(NO3)2, Pb-EDTA) was assessed in leaves of H. annus, Z. mays, L. sativa, and C. annuum exposed in hydroponic conditions [25,26,28–30]. However, those studies were performed between 2006 and 2011, when the LIBS spatial resolution was still low (typically 500 µm), and the analyzed area did not capture the whole samples, which were typically several mm2 in size. However, the main goal of these studies was to establish the total Pb content in leaves [25,26], the content of chlorophylls a and b in root and leaves [28], and morphological changes (e.g. dry and fresh weight of roots or shoots), total protein content, enzyme activity, and the content of low-molecular-mass thiols [29]. 13

The spatial Pb distribution was commonly confirmed with a complementary technique, LA-ICP-MS [28–30]. In these studies, the value of knowing the exact Pb position in relation to other data was highlighted. However, it should be mentioned that the analyzed areas were small and the sample preparation was poor [28]. Nevertheless, those pioneer studies demonstrated that LIBS is a useful complementary technique in phytotoxicity studies. The distribution of Ag was assessed in V. faba roots and H. annus leaves. Ag+ ions were shown to be fairly homogeneously distributed in the root cortex, and their effects on the root morphology – length, color, and the presence of lateral roots – were also detected [34]. In a pioneering study [27], only two laser-ablation based analytical methods (LIBS and LA-ICP-MS) were evaluated as for their ability to map the distribution of Ag in the leaves of H. anuus. Effects of a toxic heavy metal, Cd, were assessed in L. minor [35]. The plants were exposed to a CdCl2 solution at various concentrations (0 mg, 0.01 mg, 0.05 mg, 0.1 mg, 0.5 mg, 1.0 mg, 2.5 mg, 5.0 mg, 7.5 mg, 10 mg, and 15 mg CdCl2/L) for 168 hours. Toxicity parameters (growth rate and growth rate inhibition), Cd bioaccumulation via the bioaccumulation factor (BAF), and the Cd distribution in whole leaves were then analyzed with LIBS (200 µm step size). Based on the analyses, the EC50 values (final biomass inhibition, 0.08 mg Cd/L, and growth rate inhibition, 0.39 mg Cd/L) and BAF value (6607) were obtained. The study concluded that the quantitative distribution of Cd depends on its exposure concentration, whereas the spatial distribution of Cd is independent of its concentration. Also, Cd was detected with NELIBS with a spatial resolution of 300 µm in L. sativa leaves after their growing in Cd(NO3)2 solution [39]. This assessment was the first spatially resolved analysis of a targeted application of NPs (Ag NPs, 80 nm) on the plant surface, which was done to enhance the Cd signal. Cd was found to be accumulated predominantly at the leaf vein intersections rather than in the mesophyll, and predominantly in the main veins rather than in the side veins. Nevertheless, a drop of NPs formed a coffee ring effect after drying. A non-homogeneous distribution of NPs was observed. This in turn negatively influenced the disproportional enhancement of Cd signal in the NELIBS experiment and the whole semi-quantitative mapping. Crop plants (R. sativus) as well as aquatic model plants (L. minor) were exposed to the mixture of Y and Yb chlorides. In short-term toxicity tests (72 h or 168 h), toxicity endpoints (R. sativus – root and hypocotyl length; L. minor – frond area), BAFs, and translocation factors for R. sativus were assessed, and LIBS maps of Y and Yb for whole R. sativus plants and L. minor leaves were recorded at a spatial resolution of 100 µm. This study demonstrated that LIBS mapping is a useful supplementary technique in classic phytotoxicity tests and that it is especially beneficial to trophic transfer tests where knowledge of the exact position of elements in whole plants is particularly important. LIBS yielded excellent results in a study of the accumulation of Li and its diffusion in leaves of P. macrophyllus after an exposure to LiCl solution for 8–48 h [36]. The study demonstrated that Li diffuses through plant leaves via their veins (i.e., bundles of vascular tissue) and that its concentration decreases after removing the leaves from the LiCl solution. The assessment of the 2D-distribution of Cr in the leaves of O. sativa [38] focused on data evaluation (prediction models based on feature variables and global spectra) and on different sensitivities of various LIBS arrangements (SP-LIBS versus DP-LIBS). The re-heating DP-LIBS was successfully used to visualize the subcellular distribution of Cr in O. sativa leaves by using prediction models based on global spectra. 14

The studies listed above demonstrate that the elemental bioimaging can improve the precision of phytotoxicity evaluations. However, these studies were still mostly focused on the optimization of LIBS parameters, its set-up arrangement, and data evaluation. The plant bioimaging served only as “case studies” for LIBS technique. These pioneering studies should be considered as the first proof of the LIBS use in the assessment of spatial element distribution in various plant tissues. However, the research field is obviously progressing, and a widespread use of LIBS as a routine screening method is expected in the future. 3.3 Nanoparticles The production of new nanomaterials has been dramatically rising during the last decades. Prior their utilization in a wide range of applications, their diverse and possibly negative effects on organisms in all trophic levels in the environment should be considered. However, detailed data about the distribution of NPs in plants are still missing. Thus, there is a demand for a novel analytical tool providing precisely their spatial distribution. This demand on technology and instrumentation is strengthened by the necessity for a large-scale imaging of complete model organisms. Until now, LIBS was used only rarely to detect NPs in any kind of biological matrices [48]. The distribution of Ag NPs [34], CdTe NPs [35], and photon-upconversion NPs [37] was analyzed in aquatic (L. minor) and terrestrial (V. faba, R. sativus) plants after the exposure to NP dispersions of various concentrations for either 168 h (V. faba, L. minor) or 72 h (R. sativus). The first visualization of NPs in plant tissues with LIBS was published in 2017 [34]. The difference in the distribution of Ag+ ions and Ag NPs (21.7 ± 2.3 nm in diameter) in V. faba root cross-sections was detected with a resolution of 50 µm. Ag NPs were localized in the outermost layers of root sections close to the rhizodermis, whereas the distribution of ions was homogenous across the entire root sections. These data pointed out that the uptake rate of NPs was very low, which is in line with current literature [49,50]. The next study investigated the spatial elemental distribution of Cd originating from different sources, namely aquatic dispersions of two types of CdTe NPs (NPs capped with glutathione: GSH CdTe NPs with a diameter of 4–4.4 nm; CdTe NPs capped with 3-mercaptopropionic acid: MPA CdTe NPs with a diameter of 4.0–5.4 nm) and a positive control (CdCl2 solutions) [35]. The NPs were shown to be unstable in aquatic environment in direct contact with the model plant (L. minor). TEM analysis of the leaf cross-sections confirmed that NPs were not adsorbed or taken up by the plants, and 2D LIBS maps showed no differences in the distribution of Cd originating from either NPs or CdCl2. The most recent LIBS study visualized the distribution of Y and Yb originating from photonupconversion NPs (NaYF4:Yb3+, Er3+-SiO2-COOH NPs, 32.5 ± 7.9 nm in diameter) in L. minor and R. sativus [37]. The 100 µm spatial resolution for Y and Yb was shown to be sufficient for the imaging of entire plants. The occurrence of NPs in their original form as well as their uptake and translocation from roots into leaves was proven with photon up-conversion scans. The most important outcome of the study was the discovery that NPs have a significant potential for trophic transfer owing to their stability, low toxicity, and low bioaccumulation in comparison to ionic forms of Y and Yb. The speciation of detected elements (ions, NPs) should be confirmed by another analytical method, as shown in [35,37]. Methods such as TEM can achieve a much better resolution but are not capable of analyzing large-sized samples or a large number of samples [35]. On the other hand, fluorescence 15

or photon up-conversion can detect special optical properties, however, they are not useful for optically inactive NPs or unstably quenching NPs [37]. As for the current resolution limit of LIBS analysis, the value is in the order of tens to hundreds of µm. Thus, LIBS can serve only as a screening method for large samples. A screening with LIBS is able to discover interesting parts of a sample that could be further subjected to a more sensitive, detailed, and time-consuming analysis. 3.4 Organic compounds So far, only Dong research group used LIBS to image organic compounds in biotic tissues [33,39]. They studied the distribution of the pesticide chlorpyrifos by mapping Cl and P in the leaves of Z. mays directly in situ with the so-called LipsImag apparatus [33] and of A. schoenoprasum in laboratory conditions with NELIBS [39]. The in situ LIBS analysis [33] proved to be suitable for qualitative element mapping of plant leaves after the pesticide had been sprayed on the plant surface directly in the field. Instead of ablating the whole leaf in a single layer, as it is a common practice [26], leaves were ablated in several thin layers at a depth of approximately 12 µm and with a spatial resolution of 200 µm. Consequently, it was possible to compile 3D models of pesticide diffusion through maize leaves. However, only small parts of leaves were analyzed (4 × 4 mm), which could not give the information on the distribution/translocation of selected contaminants through the whole leaves/plant. In their next study, the NELIBS approach towards signal enhancement was used for a spatially resolved plant analysis [39]. Ag NPs were dropped onto the leaf surface of A. schoenoprasum in order to enhance the signal of P and Cl and make them more easily detectable. LODs which were assessed for selected wavelengths of P were found to be improved of two orders of magnitude in comparison to the LODs of classic LIBS. However, this achievement cannot be used for in situ experiments because the field application of NPs could contaminate edible crops.

16

4. Comparison of performance with other methods of elemental imaging Table 2 briefly describes the most common bioimaging methods for the assessment of elemental distribution in plants, which are based on different physical principles (X-ray-based, secondary-ionbased, and laser-based). Basic parameters such as lateral resolution, sensitivity, and the possibility of in situ analysis or quantification are noted in the table. One of their most important features is spatial resolution which varies from method to method, depending on its underlying principle of operation, and determines the possibility of application in plant analysis. The most common X-ray based methods, (SEM-EDX and PIXE) as well as those based on X-ray fluorescence (XRF, μ-XRF) and X-ray absorption (XAS, XANES, XAFS, EXAFS) have a long history in plant analysis. Detailed principles of their operation can be found elsewhere [8,9]. These techniques require mounting of plant cross-sections onto plastic foil supports instead mounting onto common glass slides. X-ray methods offer a possibility to construct 3D models from 2D maps as well as they enable in situ use and possible quantification. Their biggest disadvantage is the susceptibility to artifacts, especially from As and Se [8,9]. Secondary-ion-based methods, SIMS and nanoSIMS, use an energetic primary ion beam to remove particles from a few top atomic layers of a sample surface. Ions emitted during this bombardment, known as secondary ions, are directed into a mass spectrometer, where they are analyzed. They can provide the elemental or molecular distribution within the samples with a spatial resolution of 0.5– 10 µm. The penetration depth, which is only 0.2–10 nm, might be disadvantageous when analyzing plant cross-sections that have a width of 5–30 µm as it only reaches the top surface layer. The method allows for depth profiling and a construction of 3D models, but only in case of very small objects down to subcellular levels. The biggest advantage is a high sensitivity (>ng/g), with nanoSIMS reaching a resolution down to 50 nm [6,7]. Laser-based ablation caused by a focused thin laser pulse serves as the sampling step in several methods. In LA-ICP-MS, the ablated material is transported by the carrier gas into the ICP source of the ICP-MS, where it is ionized. The ions within a mass range of interest are separated by their massto-charge ratio in a mass analyzer (the most commonly used quadrupole) and then detected. A high sensitivity down to the ng/g range, broad spatial resolution window of 1–500 µm, and coupling to the routinely used ICP-MS method makes LA-ICP-MS one of the most often used tools in plant bioimaging [6,51,53]. If necessary, the spatial resolution can be lowered down to 0.3 µm by laser microdissection apparatuses in the LMD-ICP-MS arrangement [52]. On the contrary, LA-ICP-OES (laser ablation with inductively coupled plasma optical emission spectrometry) was used for bioimaging of plant samples (Ca, Na, and K distribution in cross-sections of tobacco leaves and stems) only once, but with a very good resolution of 15 µm [54]. Laser-ablation-based techniques have been proven as time-saving methods. Moreover, low LODs enable a simultaneous multi-element detection at relatively low costs. In comparison to other laser-ablation-based techniques, only LIBS can provide in situ quantitative analysis with a satisfactory sensitivity and optionally wide lateral resolution window.

17

Tab. 2: Comparison of X-ray based methods, secondary ion-based methods, and laser-based methods for the assessment of spatial element distribution in plant samples.

Technique X-ray-based methods Scanning electron microscopy - Energy dispersive X-ray spectroscopy Proton-induced X-ray emission X-ray fluorescence X-ray absorption

Sensitivity

In situ analysis

Quantification

Recently summarized for plant/biological samples analysis

10 µm

μg/g

No

Possible

[8]

PIXE

1 µm

μg/g

Possible

Possible

[8,9]

XRF, μ-XRF XAS (XANES, XAFS, EXAFS)

0.1–10 µm

μg/g

Possible

Possible

[8,9]

0.1–5000 µm

μg/g

Possible

Possible

[8,9]

0.5–10 µm

>ng/g

No

Difficult

[6,7]

down to 50 nm

μg/g

No

Difficult

[6,7]

>ng/g

No

Possible

[6,51,53]

μg/g

No

Possible

[6,52]

μg/g

No

Possible

[53]

μg/g

No

Possible

[54]

μg/g

Possible

Possible

[13,42,43]

Acronyms

Lateral resolution

SEM-EDX

Secondary-ion-based methods Secondary ion mass SIMS spectrometry Nanoscale secondary ion nanoSIMS mass spectrometry Laser-based methods

Laser ablation inductively coupled plasma mass LA-ICP-MS 1–500 µm spectrometry Laser microdissection inductively coupled LMD-ICP-MS 0.3–10 µm plasma mass spectrometry Matrix-assisted laser desorption/ionization MALDI-MS 10–200 µm mass spectrometry Laser ablation inductively coupled plasma optical LA-ICP-OES 15 µm emission spectrometry Laser-induced breakdown LIBS 50–750 µm spectroscopy 1 Only one research paper was published so far

1

18

5. Conclusion and environmental implications This paper reviewed original studies investigating spatial element detection in plants in the period of time from 2006 (when the first such study was published) to 2019. It demonstrated their progress by the time, importance and usability of their outcomes, and especially the employed LIBS settings and arrangements. Although LIBS is still limited by its moderate spatial resolution, sensitivity, and spatially resolved quantification, the progress has been made to overcome all these limitations. The sensitivity can be enhanced by improving the set-up (such as by adding a second laser) or by manipulating the plant sample (such as by enhancing the signal with NPs). To date, the best achieved spatial resolution has been 50 µm, which is close to the LA-ICP-MS experiments. The most common range of used spatial resolution is from 100 µm to 500 µm. The resolution is selected with respect to the sample size, information need, and is limited by employed instrumentation. However, the improvement for one order of magnitude is expected soon due to the laser and optics development. Thus, LIBS represents a vital alternative to its analytical counterparts in terms of analysis of large-scale samples with a sufficient resolution. Also, quantitative mapping has been achieved only partially so far, and the common semiquantitative analysis has been shown not to suffice for current research needs. All these problems are expected to be solved in the near future, enhancing the existing LIBS benefits such as low cost, simple instrumentation, fast multi-element screening of a large number of samples as well as of large-sized samples (typically in the range of mm2 to tens of cm2), acceptable LODs for many types of experiments, and a possibility to detect almost all elements, including light ones. Also, LIBS has been proven applicable for in situ experiments in field conditions and recently also for 3D-model compilation. In conclusion, it is particularly beneficial to use LIBS for a fast screening in common phytotoxicity tests if it is desired to obtain the information about the site of bioaccumulation, translocation, uptake routes as well as about contaminants’ trophic transfer. All this information could improve the impact of phytotoxicity research, particularly because it is critically important to understand these processes to protect the quality of global ecosystems. LIBS can predominantly serve as a fast screening method for a large number of samples or as a complementary method providing other details to the complex analysis. Moreover, LIBS could be used in the field conditions to get a preliminary look before a laborious and not trivial sample selection, collection, storing, and transfer for a laboratory analysis.

ACKNOWLEDGMENTS This research has been financially supported by the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601). Also, this work was carried out with the support of CEITEC Nano Research Infrastructure (MEYS CR, 2016–2019) and CEITEC Nano+ project, ID CZ.02.1.01/0.0/0.0/16_013/0001728.

Conflicts of interest There are no conflicts to declare. 19

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24

HIGHLIGHTS Means to implement LIBS into the toxicological routine are discussed. Benefits and drawbacks of LIBS are put into context with respect to other analytical techniques. Plant exposure to compounds and nanoparticles follows by plant preparation prior LIBS analysis is investigated in detail. Extending information on the influence of nanoparticles on plant organisms gained from LIBS analysis are showed. Imaging of nanoparticles in plant tissue, a novel trend in laser-induced breakdown spectroscopy is reviewed.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: