**4. Elemental distribution imaging via LA-ICP-MS and LIBS techniques**

Images are present in daily life of the population in different contexts, such as through the image created by the eyes, enabling observation of the environmental around us; through pictures and photographs that record different personal or historical moments; and also, via magnetic resonance imaging (MRI) or X-ray computed tomography (CT) which contribute to medical diagnostics. In the chemistry context, imaging is a process that transforms the spectral information of atoms and molecules present in the solid sample surface, in a high resolution image through the application of powerful spectral techniques as LA-ICP-MS, LIBS, Raman [41], X-Ray Spectroscopy [42] and Secondary Ion Mass Spectrometry (SIMS) [43].

For the LA-ICP-MS and LIBS techniques, the imaging process occurs through the applications of laser pulses directed at specific regions of the sample surface (*x,y* coordinate), via point by point or continuous lines performing the chemical measurements and obtaining spectral information of the region of interest [22, 44]. In this case, a document (.txt or .log format, for example) is generated for each point or a line containing the signal intensity data of all the elements measured [45]. Due to the complexity and a large number of data obtained, appropriate software for data processing is required to separate the information and generate a data matrix, thus allowing the generation of a final image for each element. A two (2D) or a three (3D) dimensions image can be created for each element in the sample. The 2D image is equivalent just to the surface image and the signal intensity or concentration of the analyte, obtained by the conventional method describe previously. The 3D image can be obtained in two ways, i) analyzing and combining each layer of the sample (volume reconstruction of several 2D images), ii) repeated pulses of laser in the same region, allowing in-depth elemental imaging [46]. The scheme of the imaging process *via* LA-ICP-MS or LIBS can be seen in **Figure 2**.

There are different software for data processing and generation of chemical images, such as Microsoft Excel, LA-iMageS [47], and MATLAB [48]. In the case of LIBS imaging, the use of chemometrics tools has been a great ally in the data treatment due to the complexity of the emission spectra data. Principal component analysis (PCA) and partial least squares (PLS) are commonly used, and also can

#### **Figure 2.**

*Steps of the imaging process via LA-ICP-MS and LIBS. Final 3D and 2D nanoparticles images by Gimenez et al. [46], reproduced with permission of Springer Nature.*

generate hyperspectral images [22, 49]. The final images are formed by a matrix of pixels, represented by I(*x,y,z*), in which *x,y* are the sample coordinates and *z*, the spectral information. Therefore, the number of pixels that will form the final image will be directly related to the amount of information, image quality, and the spatial resolution that the imaging process can provide.

The spatial resolution is the ability of the imaging method to distinguish two points in the sample surface, also mentioned as lateral resolution. It is correlated to the spot size and line scan direction, the x- and y-resolution. The x-resolution (μm) is related to the scan direction and the pixel size of each data set, obtained by multiplication of the scan speed and the acquisition time. The y-resolution (μm) is correlated to the distance between the lines. Thus, the spatial resolution and, consequently, the pixel size, is a result of the laser spot size, scan speed, acquisition time, and the distance between the measured lines or points. These parameters vary according to the application and optimized instrumental parameters. It is important to mention that LA-ICP-MS and LIBS are considered high spatial resolution techniques [48, 50, 51].

The imaging process has the advantage of accessing spatial and distribution information of chemical species that would not be possible using the conventional method of analysis, as atomic emission spectroscopy (AES) and inductively coupled plasma mass spectrometry (ICP-MS). These techniques usually require sample preparation steps, as decomposition and/or extraction, that cause destruction of the sample and, consequently, of spatial information. One of the major limitations of imaging via LA-ICP-MS and LIBS is the long data acquisition time, varying according to the size of the analyzed sample, scan speed, and laser parameters, as well as making the whole system result in representative images of the system in evaluation [22, 44]. Quantitative imaging based on LIBS and LA-ICP-MS is still a challenge and a controversial topic between researchers. Most methodologies are considered just qualitative or semi-quantitative, but in the literature, it can be observed different studies that aim to developed calibrations and mathematical strategies to transform the relative intensity information into a quantitative image [46, 52]. The applicability of the LA-ICP-MS and LIBS imaging can provide spatial chemical information to solve problems in different areas of science as demonstrated in the next section.

#### **4.1 Applications of elemental distribution imaging via LA-ICP-MS and LIBS**

Biomedical researches and clinical diagnosis are some of the most important areas of chemical imaging applications because is necessary to know the chemical specie involved in different diseases and the specific localization in human tissues, thus allowing specific drug development. Recent reviews of LIBS [22] and LA-ICP-MS [53] imaging applied in medical science can be found in the literature.

Moncayo *et al.* applied LIBS imaging of human paraffin-embedded skin samples as complementary biopsy with conventional histopathology sample preparation to detect the differences of the spatial distribution of P, Mg, Na, Ca, Zn and Fe in healthy and malignant skin tissues of cutaneous metastasis of melanoma, Merkellcell carcinoma (MCC) and squamous cell carcinoma (SCC). In this study, two lens-fibred system and two spectrometers were used to realize the simultaneous detection of all elements. The high-resolution images were obtained using laser shots with 50 μm of step size and an acquisition time of 3 hours for each sample with, approximately, 3cm2 of the tumor area. As imaging results, high levels of P were observed in all skins in the tumors areas; high levels of Ca and Zn were noticed closest to the tumor and decrease in the areas away from the tumor in the MCC; a gradient level of Ca and Mg in metastatic melanoma and Na, Mg and Zn in

**151**

**Figure 3.**

*silver at the end of cultivation is 12 mg kg−1), using 13C+*

*Laser Chemical Elemental Analysis: From Total to Images*

SCC were observed in the left area of the tumor, allowed proper identification and

Studies involving NPs has increased in the last years because of their versatility of applications, such as in medical science (drug delivery, label method or immunotherapy) and environmental (nutrient or toxic transport) [55–57]. Imaging the distribution of Ag, Cu, and Mn in soybean leaves cultivated in the presence or the absence of AgNPs (40 nm average size) and also using AgNO3 indicated Ag poor translocation to leaves. Additionally, the homeostasis of Mn and Cu is highly affected in plants cultivated in the presence of AgNO3 compared to those cultivated in the presence of AgNPs (**Figure 3**) [56]. Additionally, the study of bio-distribution of NPs is a challenge because of their small size (< 100 nm), so high spatial resolution imaging techniques present as promising assessment tools to evaluation at the

*Ag, Cu and Mn distributions in soybean leaves for the groups (A) control, (B) AgNO3 and (C) AgNPs (total* 

*of this figure [56] - reproduced by permission of The Royal Society of Chemistry [56].*

 *as the IS. Each real soybean leaf is presented on the top* 

discrimination of the three different skins analyzed by LIBS imaging [54]. Applying quantitative LA-ICP-MS imaging using line scan mode, the spot diameter of 10 μm, 20 μm s−1 scan speed, and matrix-matched calibration, Crone *et al.* could evaluate the ineffectiveness of Pt drug-based in bone metastasis treatment in a mouse tibia samples, in which are generally considered an unsatisfactory and scarce treatment. According to the results, there is no efficient transport of the drug into the bones, in which presented lowest concentration of Pt and, the highest concentration were found on the outside of the bone samples, confirming the

hypothesis that the Pt drug did not penetrate the bones [55].

*DOI: http://dx.doi.org/10.5772/intechopen.94385*

cellular level and macroscales.

#### *Laser Chemical Elemental Analysis: From Total to Images DOI: http://dx.doi.org/10.5772/intechopen.94385*

*Practical Applications of Laser Ablation*

resolution techniques [48, 50, 51].

resolution that the imaging process can provide.

generate hyperspectral images [22, 49]. The final images are formed by a matrix of pixels, represented by I(*x,y,z*), in which *x,y* are the sample coordinates and *z*, the spectral information. Therefore, the number of pixels that will form the final image will be directly related to the amount of information, image quality, and the spatial

The spatial resolution is the ability of the imaging method to distinguish two points in the sample surface, also mentioned as lateral resolution. It is correlated to the spot size and line scan direction, the x- and y-resolution. The x-resolution (μm) is related to the scan direction and the pixel size of each data set, obtained by multiplication of the scan speed and the acquisition time. The y-resolution (μm) is correlated to the distance between the lines. Thus, the spatial resolution and, consequently, the pixel size, is a result of the laser spot size, scan speed, acquisition time, and the distance between the measured lines or points. These parameters vary according to the application and optimized instrumental parameters. It is important to mention that LA-ICP-MS and LIBS are considered high spatial

The imaging process has the advantage of accessing spatial and distribution information of chemical species that would not be possible using the conventional method of analysis, as atomic emission spectroscopy (AES) and inductively coupled plasma mass spectrometry (ICP-MS). These techniques usually require sample preparation steps, as decomposition and/or extraction, that cause destruction of the sample and, consequently, of spatial information. One of the major limitations of imaging via LA-ICP-MS and LIBS is the long data acquisition time, varying according to the size of the analyzed sample, scan speed, and laser parameters, as well as making the whole system result in representative images of the system in evaluation [22, 44]. Quantitative imaging based on LIBS and LA-ICP-MS is still a challenge and a controversial topic between researchers. Most methodologies are considered just qualitative or semi-quantitative, but in the literature, it can be observed different studies that aim to developed calibrations and mathematical strategies to transform the relative intensity information into a quantitative image [46, 52]. The applicability of the LA-ICP-MS and LIBS imaging can provide spatial chemical information to solve problems in different areas of science as demonstrated in the next section.

**4.1 Applications of elemental distribution imaging via LA-ICP-MS and LIBS**

P were observed in all skins in the tumors areas; high levels of Ca and Zn were noticed closest to the tumor and decrease in the areas away from the tumor in the MCC; a gradient level of Ca and Mg in metastatic melanoma and Na, Mg and Zn in

Biomedical researches and clinical diagnosis are some of the most important areas of chemical imaging applications because is necessary to know the chemical specie involved in different diseases and the specific localization in human tissues, thus allowing specific drug development. Recent reviews of LIBS [22] and LA-ICP-MS [53] imaging applied in medical science can be found in the literature. Moncayo *et al.* applied LIBS imaging of human paraffin-embedded skin samples as complementary biopsy with conventional histopathology sample preparation to detect the differences of the spatial distribution of P, Mg, Na, Ca, Zn and Fe in healthy and malignant skin tissues of cutaneous metastasis of melanoma, Merkellcell carcinoma (MCC) and squamous cell carcinoma (SCC). In this study, two lens-fibred system and two spectrometers were used to realize the simultaneous detection of all elements. The high-resolution images were obtained using laser shots with 50 μm of step size and an acquisition time of 3 hours for each sample

of the tumor area. As imaging results, high levels of

**150**

with, approximately, 3cm2

SCC were observed in the left area of the tumor, allowed proper identification and discrimination of the three different skins analyzed by LIBS imaging [54].

Applying quantitative LA-ICP-MS imaging using line scan mode, the spot diameter of 10 μm, 20 μm s−1 scan speed, and matrix-matched calibration, Crone *et al.* could evaluate the ineffectiveness of Pt drug-based in bone metastasis treatment in a mouse tibia samples, in which are generally considered an unsatisfactory and scarce treatment. According to the results, there is no efficient transport of the drug into the bones, in which presented lowest concentration of Pt and, the highest concentration were found on the outside of the bone samples, confirming the hypothesis that the Pt drug did not penetrate the bones [55].

Studies involving NPs has increased in the last years because of their versatility of applications, such as in medical science (drug delivery, label method or immunotherapy) and environmental (nutrient or toxic transport) [55–57]. Imaging the distribution of Ag, Cu, and Mn in soybean leaves cultivated in the presence or the absence of AgNPs (40 nm average size) and also using AgNO3 indicated Ag poor translocation to leaves. Additionally, the homeostasis of Mn and Cu is highly affected in plants cultivated in the presence of AgNO3 compared to those cultivated in the presence of AgNPs (**Figure 3**) [56]. Additionally, the study of bio-distribution of NPs is a challenge because of their small size (< 100 nm), so high spatial resolution imaging techniques present as promising assessment tools to evaluation at the cellular level and macroscales.

#### **Figure 3.**

*Ag, Cu and Mn distributions in soybean leaves for the groups (A) control, (B) AgNO3 and (C) AgNPs (total silver at the end of cultivation is 12 mg kg−1), using 13C+ as the IS. Each real soybean leaf is presented on the top of this figure [56] - reproduced by permission of The Royal Society of Chemistry [56].*

Krajcarová *et al.* used double-pulse LIBS image mapping to verify the spatial distribution of Ag<sup>+</sup> and AgNPs in a small root cross-section of *Vicia faba* and their differences in transport efficiency. Ag<sup>+</sup> ions are toxic for plant tissues and the AgNPs are the most used as an antimicrobial agent in many products applied in plants. However, the later can interfere in the growth and biomass production if absorbed by the roots of the plants. Using a 50 μm of image resolution, the authors observed a clear difference between the localization of Ag<sup>+</sup> ions and AgNPs. AgNPs were found in just one part of root layers, close to the rhizodermis, and had no significant visual effects on the roots, just reduced the lateral root formation. Meanwhile, Ag<sup>+</sup> presented higher signal intensities and homogeneous distribution in the root cortex and caused reduced root lengths, darker color, and absence of lateral roots [58].

LA-ICP-MS imaging and NPs was used by Cruz-Alonso *et al.* to evaluate molecular distribution and quantification in human retina sections of 4 postmortem donors. Gold nanoparticles were applied as antibody-conjugated nanoclusters (Ab-AuNCs) to identify metallothioneins (MTs), which are protective to the neural retinal cells against oxidative stress. The LA-ICP-MS quantitative imaging (expressed in ng of MT g−1) was applied in line scan mode with 10 μm of spot diameter and a matrix-matched analytical curve was used as a calibration strategy. As the sample is a biological tissue, the authors replaced the commercial ablation chamber by an in-house chamber with a reduced internal volume and cell temperature constant at −20°C, keeping the sample integrity. As results, the Ab-AuNCs allowed observed that the MTs present distribution in some principals retinas layers, known inner and outer nuclear layers and ganglion cells layers, as well as a present different concentration between the 4 postmortem donors, which is correlated with the biological diversity characteristics of each individual patient [52].

Different applications of imaging process applied to environmental studies [22], and translocation and accumulation of metals in plant tissues [59]. Paleoclimate and geological researches developed important studies in environmental science aiming to understand climate and environmental impacts using tree rings and speleothems as natural archive samples. These samples present high temporal resolution, allowing temporal reconstruction because changes in environmental or climate conditions can interfere on growth periods and elemental composition. Locosseli *et al.* demonstrated with LA-ICP-MS imaging that the decrease of Pb through the tree rings of *Tipuana tipu* (Fabaceae) could be correlated to the decrease of Pb concentration in gasoline of São Paulo (Brazil). In this study, high-resolution scan mode with x-resolution of 31.5 μm, scan speed varying of 60–100 μm s−1, and LA-iMageS software was used [60].

A limitation in environmental studies is the small sample size that is allowed in the ablation chamber used in LA-ICP-MS, limiting experimental sampling. As an advantage of LIBS imaging, the ablation chamber does not be needed to be closed. Recently, Cáceres *et al.* reported the development of a method that enables the create megapixels elemental image of large speleothems (25 cm long) and coral (8 cm long) surfaces by fast and high-resolution LIBS imaging. Approximately 360,000 pixels hour−1 were obtained in a 10 μm of lateral resolution, 100 Hz (100 pixels s−1) as operation speed, and 8 hours per 2D imaging created with LasMap software. It allowed the visualization of fibers and specific regions of coral with the Mg/Ca, Sr/Ca and Na/Ca images, and according to the authors, the speleothems images demonstrated that compositional variations of Sr/Ca and Mg/Ca are not random but they result of variations of paleoenvironmental proxies [61].

As previously mentioned, LA-ICP-MS and LIBS can have the same initial laser system (**Figure 1**), which is a multimodal method in the imaging process.

**153**

*Laser Chemical Elemental Analysis: From Total to Images*

This approach was applied for Bonta *et al.* to create an elemental mapping of biological tissues with a tumor of human malignant pleural mesothelioma (MPM) (**Figure 4**). Using a line scan with 40 μm of spot diameter, 40 μm of lateral resolution, 80 μm s−1 of scan speed and the software ImageLab to create the images, the authors observed that O, P, Zn, and Cu exhibit similar heterogeneous spatial distribution in the tissue, with high signal intensity in the tumor region, correlated with high number of activated proteins and their cofactors in this area. Different elements were detected in the tumor area, the presence of Pt, originated from common anti-cancer drug cisplatin, was detected in the healthy area, but not in the tumor's region [62]. This information would not be observed using a conventional

*Multimodal images of human tumor. Adapted from Bonta et al. [62] with permission from The Royal Society* 

Remarkable research and developments in laser technology and atomic spectrometry have promoted LIBS and LA-ICP-MS techniques to the height of its maturity in terms of both instrumentation and applications. Sensitivity, precision, and accuracy have continuously been improved, and obviously dependent on the experimental configurations used (type of laser, spectrometers, and detectors). These techniques allow the direct analysis of solids which is a significant advantage for the analysis of difficult-to-digest materials. High spatial resolution capacity allows for detailed information of the sample surface composition. All these features result in promising solutions for studies which the spatially resolved elemental information are required to provide a better understanding of the sample with significant advantages over conventional bulk analyses, such as in medical, environmental and technological science. Because LIBS present simpler instrumentation and an interesting trend is the portable instruments particularly attractive for in situ applications such as in agriculture and environmental analysis. Still, some researches focus on quantitative developments and calibration strategies to overcome remaining drawbacks such as the lack of CRMs, matrix effects and fractionation as well as multimodal system and isotopic analysis by LA-ICP-MS. At this stage of development, we may point out that LA-ICP-MS and LIBS have complementary analytical performances and may be considered as attractive alternatives for the analysis of both bulk samples and elemental imaging distribution.

*DOI: http://dx.doi.org/10.5772/intechopen.94385*

method of analysis via decomposition.

**5. Conclusions**

**Figure 4.**

*of Chemistry.*

#### **Figure 4.**

*Practical Applications of Laser Ablation*

differences in transport efficiency. Ag<sup>+</sup>

distribution of Ag<sup>+</sup>

Meanwhile, Ag<sup>+</sup>

lateral roots [58].

Krajcarová *et al.* used double-pulse LIBS image mapping to verify the spatial

AgNPs are the most used as an antimicrobial agent in many products applied in plants. However, the later can interfere in the growth and biomass production if absorbed by the roots of the plants. Using a 50 μm of image resolution, the authors

were found in just one part of root layers, close to the rhizodermis, and had no significant visual effects on the roots, just reduced the lateral root formation.

in the root cortex and caused reduced root lengths, darker color, and absence of

LA-ICP-MS imaging and NPs was used by Cruz-Alonso *et al.* to evaluate molecular distribution and quantification in human retina sections of 4 postmortem donors. Gold nanoparticles were applied as antibody-conjugated nanoclusters (Ab-AuNCs) to identify metallothioneins (MTs), which are protective to the neural retinal cells against oxidative stress. The LA-ICP-MS quantitative imaging (expressed in ng of MT g−1) was applied in line scan mode with 10 μm of spot diameter and a matrix-matched analytical curve was used as a calibration strategy. As the sample is a biological tissue, the authors replaced the commercial ablation chamber by an in-house chamber with a reduced internal volume and cell temperature constant at −20°C, keeping the sample integrity. As results, the Ab-AuNCs allowed observed that the MTs present distribution in some principals retinas layers, known inner and outer nuclear layers and ganglion cells layers, as well as a present different concentration between the 4 postmortem donors, which is correlated with the biological diversity characteristics of each individual patient [52]. Different applications of imaging process applied to environmental studies [22], and translocation and accumulation of metals in plant tissues [59]. Paleoclimate and geological researches developed important studies in environmental science aiming to understand climate and environmental impacts using tree rings and speleothems as natural archive samples. These samples present high temporal resolution, allowing temporal reconstruction because changes in environmental or climate conditions can interfere on growth periods and elemental composition. Locosseli *et al.* demonstrated with LA-ICP-MS imaging that the decrease of Pb through the tree rings of *Tipuana tipu* (Fabaceae) could be correlated to the decrease of Pb concentration in gasoline of São Paulo (Brazil). In this study, high-resolution scan mode with x-resolution of 31.5 μm, scan speed varying of

observed a clear difference between the localization of Ag<sup>+</sup>

60–100 μm s−1, and LA-iMageS software was used [60].

but they result of variations of paleoenvironmental proxies [61].

As previously mentioned, LA-ICP-MS and LIBS can have the same initial laser system (**Figure 1**), which is a multimodal method in the imaging process.

A limitation in environmental studies is the small sample size that is allowed in the ablation chamber used in LA-ICP-MS, limiting experimental sampling. As an advantage of LIBS imaging, the ablation chamber does not be needed to be closed. Recently, Cáceres *et al.* reported the development of a method that enables the create megapixels elemental image of large speleothems (25 cm long) and coral (8 cm long) surfaces by fast and high-resolution LIBS imaging. Approximately 360,000 pixels hour−1 were obtained in a 10 μm of lateral resolution, 100 Hz (100 pixels s−1) as operation speed, and 8 hours per 2D imaging created with LasMap software. It allowed the visualization of fibers and specific regions of coral with the Mg/Ca, Sr/Ca and Na/Ca images, and according to the authors, the speleothems images demonstrated that compositional variations of Sr/Ca and Mg/Ca are not random

and AgNPs in a small root cross-section of *Vicia faba* and their

presented higher signal intensities and homogeneous distribution

ions are toxic for plant tissues and the

ions and AgNPs. AgNPs

**152**

*Multimodal images of human tumor. Adapted from Bonta et al. [62] with permission from The Royal Society of Chemistry.*

This approach was applied for Bonta *et al.* to create an elemental mapping of biological tissues with a tumor of human malignant pleural mesothelioma (MPM) (**Figure 4**).

Using a line scan with 40 μm of spot diameter, 40 μm of lateral resolution, 80 μm s−1 of scan speed and the software ImageLab to create the images, the authors observed that O, P, Zn, and Cu exhibit similar heterogeneous spatial distribution in the tissue, with high signal intensity in the tumor region, correlated with high number of activated proteins and their cofactors in this area. Different elements were detected in the tumor area, the presence of Pt, originated from common anti-cancer drug cisplatin, was detected in the healthy area, but not in the tumor's region [62]. This information would not be observed using a conventional method of analysis via decomposition.

## **5. Conclusions**

Remarkable research and developments in laser technology and atomic spectrometry have promoted LIBS and LA-ICP-MS techniques to the height of its maturity in terms of both instrumentation and applications. Sensitivity, precision, and accuracy have continuously been improved, and obviously dependent on the experimental configurations used (type of laser, spectrometers, and detectors). These techniques allow the direct analysis of solids which is a significant advantage for the analysis of difficult-to-digest materials. High spatial resolution capacity allows for detailed information of the sample surface composition. All these features result in promising solutions for studies which the spatially resolved elemental information are required to provide a better understanding of the sample with significant advantages over conventional bulk analyses, such as in medical, environmental and technological science. Because LIBS present simpler instrumentation and an interesting trend is the portable instruments particularly attractive for in situ applications such as in agriculture and environmental analysis. Still, some researches focus on quantitative developments and calibration strategies to overcome remaining drawbacks such as the lack of CRMs, matrix effects and fractionation as well as multimodal system and isotopic analysis by LA-ICP-MS.

At this stage of development, we may point out that LA-ICP-MS and LIBS have complementary analytical performances and may be considered as attractive alternatives for the analysis of both bulk samples and elemental imaging distribution.
