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Article

Application of a Multi-Technique Approach to the Identification of Mineral Polymorphs in Histological Samples: A Case of Combined Use of SEM/EDS and Micro-Raman Spectroscopy

by
Alessandro Croce
1,2,*,
Donata Bellis
3,
Caterina Rinaudo
2,
Laura Cagna
2,
Giorgio Gatti
4,
Annalisa Roveta
1,
Marinella Bertolotti
1 and
Antonio Maconi
5
1
SSD Research Laboratories, Research Training Innovation Infrastructure, Research and Innovation Department (DAIRI), Azienda Ospedaliero-Universitaria SS. Antonio e Biagio e Cesare Arrigo, Via Venezia 16, 15121 Alessandria, Italy
2
Department of Science and Technological Innovation, University of Eastern Piedmont, Viale T. Michel 11, 15121 Alessandria, Italy
3
Centre “G. Scansetti” Via Pietro Giuria 9, 10100 Torino, Italy
4
Department for Sustainable Development and Ecological Transition, Piazza S. Eusebio 5, 13100 Vercelli, Italy
5
Research Training Innovation Infrastructure, Research and Innovation Department (DAIRI), Azienda Ospedaliero-Universitaria SS. Antonio e Biagio e Cesare Arrigo, Via Venezia 16, 15121 Alessandria, Italy
*
Author to whom correspondence should be addressed.
Minerals 2024, 14(7), 633; https://doi.org/10.3390/min14070633
Submission received: 24 May 2024 / Revised: 14 June 2024 / Accepted: 19 June 2024 / Published: 21 June 2024
(This article belongs to the Section Crystallography and Physical Chemistry of Minerals & Nanominerals)
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Abstract

In the last few years, an increasing interest has developed regarding the application of different techniques for the identification of pollutants inside the tissues deriving from patients affected by benign or neoplastic diseases. Particular attention was paid to neoplasia linked to particular exposures, e.g., heavy metals, carbon dusts, silica, asbestos. As regards the last pollutant, a wide body of scientific literature has been collected, considering the severe effects caused by mineral fibers on human health. Optical and electronic microscopies were widely applied to identify the fibers in respiratory and extra-respiratory organs to detect the minerals and to link their presence to an exposure source and to understand their role in cancer development. The main advantage of electron microscopy lies in the possibility of coupling the microscopes with energy dispersive spectrometers and also collecting data on the elemental composition of various inorganic phases. In term of sample preparation and time of analysis, the most utilized microscope technique is Scanning Electron Microscopy with an annexed energy dispersive spectrometer (SEM/EDS), allowing for the morphological and chemical characterization of the observed particles/fibers. Moreover, this technique is envisaged by Italian Law for asbestos identification in air and bulk samples. On the other hand, this technique does not allow a reliable identification of the mineral phase in the case of polymorphs with the same chemical formula but different crystal structures. In this work, the coupling of a spectroscopical technique—micro-Raman spectroscopy—to SEM/EDS is proposed for a sure phase identification of particles, showing EDS spectra with ambiguous phase identification, observed in samples of tissues from patients affected by colorectal cancer and living in an asbestos-polluted area. In these tissues, different particles with EDS spectra that do not allow a sure identification of the phase—in particular calcium-rich particles and titanium oxides—were successively analyzed by micro-Raman spectroscopy. Thanks to this last technique, it was possible to ascribe the mineral phases associated to these particles to “aragonite” (a calcium carbonate polymorph) and to “anatase” (a Ti dioxide polymorph).

1. Introduction

The improvement of the techniques and methodologies applied to the characterization of mineral samples—and not only them—has shown a fast improvement during the XX and XXI Centuries under different points of view, e.g., imaging, chemical characterization, bulk characterization. For instance, concerning the Scanning Electron Microscope (SEM), the first prototype was built in 1935 by Knoll, and the first SEM was developed only in 1942 by Zworykin et al. [1]. The real improvement of the technique started during the Sixties of the XX Century. Since that time, SEM instruments have enhanced more and more with the different technological discoveries and applications—e.g., the development of magnetic lenses with higher performances, the possibility of working under environmental pressures, and the improvement of informatics—up to today’s equipment. In fact, nowadays it is possible to obtain different kinds of information and manipulation can be not necessary for sample characterization [2]. Moreover, different types of detectors were developed over the years and they can be coupled in the same instrument, such as an Everhart–Thornley Detector (ETD), a Circular Backscatter Detector (CBS), or a Gaseous Analytical Detector (GAD). In SEM equipment, an important improvement was the development of Energy Dispersive Spectrometers (EDS), which allow for combining chemical information with morphological information [3].
Notwithstanding these technological enhancements, there are even now some cases where it is not possible to obtain sure information about the studied materials considering chemical characteristics. An example is represented by polymorph mineral phases. These are inorganic materials showing a different crystal structure and the same—or very close—chemical compositions. Among them, minerals belonging to serpentine phases often represent a problem in SEM/EDS studies in the field of environmental and health sciences. In fact, there are three minerals—chrysotile, antigorite, and lizardite—which are characterized by a very close theoretical chemical formula—Mg3Si2O5(OH)4—with very limited diadochy [4]. Considering the morphological characteristics, lizardite is well identified thanks to its platy-like crystals, whereas the distinction between chrysotile and antigorite is often difficult because they both crystallize in elongated fibrous form [5,6]. Nevertheless, only chrysotile is defined as an “asbestos” phase by the Law, which is a problem in medico–legal debates [7,8]. Different phases might possess similar chemical composition and different structures from a mineralogical point of view: for example, we cite calcium carbonate phases (calcite—vaterite—aragonite) [9], oxides (e.g., titanium dioxides—rutile, brookite, and anatase) [10], silica (e.g., quartz, cristobalite, and coesite) [11], and carbon (graphite and diamond) [12].
Sometimes the distinction among the polymorphs might be obvious considering other characteristics (e.g., their morphologies), but in other cases, it is necessary to combine an additional technique to ascertain the real mineral phase associated to the particles/fibers, especially when SEM/EDS is applied to collect only qualitative chemical data [13]. One of these techniques is Raman spectroscopy.
This instrumentation has also experienced rapid enhancement during the last decades, thanks to the technological improvements carried out during this period. The Raman effect—the name derived from the surname of the Indian Professor Chandrasekhara Venkata Raman who discovered it—was theorized and discovered during the Twenties of the XX Century but it saw its highest improvements with the development of laser sources in the Sixties. Nowadays, this technique may be applied to a wide range of materials, thanks to the possibility of obtaining vibrational spectra that are like “fingerprints” of the analyzed samples [14,15], which are very useful for polymorph phase identification. Moreover, the spectroscope can be coupled with optical microscopes (the so-called micro-Raman spectroscopy), giving both images and spectra of the analyzed materials. Lastly, but no less important, it is possible to carry out analyses on the samples carrying out only little or no manipulations on both organic and inorganic phases [2,16,17,18,19].
This last possibility is very important because it allows for analysis of the same samples under both SEM/EDS and micro-Raman spectroscopes, obtaining complementary information about the fibers/particles of interest. This is very important especially when a polymorph is found using the first technique [20].
In this work, an example of the importance of coupling these two techniques on samples deriving from patients affected by colon–rectum carcinoma (CRC) and those living in an asbestos-polluted area is reported. Particular attention was focused on different polymorph phases to best evaluate the mineral phases contained inside the organ. The original aim of the project was the characterization of asbestos fibers inside the tissues of 20 patients affected by colorectal cancer (CRC) and living in an area where an Eternit factory was working until 1986 [21]. SEM/EDS and Optical Microscopy (OM) were used with the objective of characterizing and quantifying the asbestos fibers inside the organs. SEM/EDS is one of the techniques indicated for asbestos phase identification, whereas Raman spectroscopy is not regulated by Italian Law. The study was funded by the CRT Foundation (Cassa di Risparmio di Torino, Turin, Italy). Nevertheless, other inorganic particles were detected inside the biological media [22], and in some cases, they were not fully recognizable from a mineralogical point of view. In the current paper, analyses carried out by SEM/EDS and micro-Raman spectroscopy to best characterize the mineral particles will be presented, mainly in the case of polymorph mineral phases.

2. Materials and Methods

CRC tissues were sampled from five patients affected by this disease who underwent surgery at the Santo Spirito Hospital of Casale Monferrato (Piedmont, Italy). The study was approved by the Institutional Review Board of the Azienda Ospedaliero-Universitaria SS. Antonio e Biagio e Cesare Arrigo of Alessandria in July 2020.
Three pieces of biological samples were collected from healthy, neoplastic, and “bridge” areas and fixed in formalin, then digested and prepared as reported in [23]. Digestion procedure was carried out by removing about 0.5 g of colon tissues using ~30 mL of sodium hypochlorite (NaClO) heated to 60 °C. This solution was then filtered on a polycarbonate filter (with a porosity of 0.2 μm and a diameter of 25 mm) to collect the inorganic material. The solution was diluted using MilliQ water heated at 60 °C to reduce the precipitation of NaCl during the filtration process. Starting from the same tissues, paraffin blocks were prepared in order to successively cut three 5 μm-thick histological sections for each sample. One of the three sections was then stained using hematoxylin and eosin (H&E), whereas two sections were left “white”.
SEM used for this study was an E-SEM Quanta 200 (FEI Company, Hillsboro, OR, USA), equipped with an EDS (EDAX, Mahwah, NJ, USA) spectrometer. SEM/EDS analyses were carried out on the inorganic residues deposited on polycarbonate filters after tissue digestion. The operative conditions were the same as reported in [23]. The samples were observed using the backscattered detector (BSD) in order to recognize the inorganic particles/fibers on the basis of their higher mean atomic number (Z), with respect to the carbonaceous substrate of the filter. EDS spectra were acquired from the characterized inorganic phases and processed using GENESIS software (version 3.6). The obtained spectra (and compositional numeric values) must be considered only as qualitative information (and the extracted numerical values as semi-quantitative). The collected analyses were compared with spectra obtained by the same instrument on pure phases and using spectra from scientific references.
Raman spectra and maps were collected using a JobinYvon Evolution spectrometer (HORIBA JobinYvon, Paris, France), equipped with a Nd:YAG laser source with a wavelength of 532 nm. Autocalibration of the instrument was carried out before every experimental session, using the ~520.6 cm−1 band of a Si standard. Micro-Raman spectroscopy was carried out on digested tissues and on white histological sections fixed in paraffin. No other particular manipulation was necessary. The spectra were acquired by focusing the 532 nm green laser using the 80× objective of the microscope on the observed particles/fibers. The full power of the excitation source was reduced to 3.2% using an opportune filter (the measured laser power on the sample was about 50 μW). In order to evaluate both inorganic and organic components, each spectrum was collected acquiring 2 accumulations of 100 s each in the 4000–100 cm−1 spectral range. The spectra were processed by Origin 6.0 software. Also in this case, the obtained results were compared with internal reference spectra, dedicated softwares (KnowItAll version 2021, Crystalsleuth version 1.0), and literature data.

3. Results

During the SEM/EDS observations, different particles and bundles of fibers were observed and morphologically and chemically characterized. Their shapes were various, both fibrous and spherical (isodiametric), but considering their dimensional features and chemical compositions, they were not classifiable as “asbestos” breathable fibers: in fact, asbestos characterization was the principal goal of this project. It must be highlighted that in addition to the chemical composition ascribable to the asbestos phases (five amphibole and one serpentine phase) [19], dimensional parameters (length, diameter, length/diameter ratio) must also be respected to count the elongated particles as regulated “breathable” fibers [2].
Remembering that asbestos bundles of fibers show a characteristic tendency to split into nanometric-sized fibrils along their elongation axis, in one of the analyzed cases, different particular morphologies were observed and classified (Figure 1). In these examples, it is evident that a lot of micro- and nanofibers form the bundles. Without a chemical evaluation, these morphologies might be classified as “asbestos”, but the EDS spectra collected on them (an example is reported in Figure 1f) showed the element “calcium” as the main component. Nevertheless, in these features, Si and Mg were also detected, sometimes with a magnesium peak higher than the one related to silicon, which is the peculiar characteristic of EDS spectra ascribed to serpentine phases [23] to which chrysotile, an asbestos phase, belongs.
In order to verify the presence of serpentine phases within the materials analyzed via SEM/EDS, spectra were recorded on the same particles using micro-Raman spectroscopy. An example of the spectrum obtained on these bundles of fibers is reported in Figure 2. The band relative to symmetric stretching of the Si-Ob-Si bonds, which lies at about 690 cm−1 in chrysotile [4,5,19], was considered to detect its presence and it was never detected in the recorded spectra (as shown by the red circle in the box of Figure 2a).
As can be seen from the spectrum reported in Figure 2a, in the 1200–100 cm−1 spectral range, a series of bands located at 1084, 204, and 151 cm−1 and a weak broadened band detected at 259 cm−1 can be observed. These bands are ascribed to the polymorph calcium carbonate “aragonite”. In fact, the band at 1084 cm−1 is attributable to the ν1(CO32−) symmetric stretching mode [24,25,26], whereas the band at 151 cm−1 is ascribed to the libration around the 0Y axis and the band at 204 cm−1 is related to the one on the 0X axis [26]. The band at 259 cm−1 is attributable to this carbonate phase [26]. However, it is not possible to clearly observe the band related to antisymmetric bending at about 705 cm−1 because a band related to the presence of the polycarbonate filter substrate falls in that position [27], even using a fitting procedure. To be sure of the correctness of the interpretation, additional particles were characterized narrowing the confocal hole of the spectroscope from 200 to 100 μm, which allows for better detection of the carbonate phase signal (Figure 2b).
Particles that generally occur as aggregates were detected in some samples with isodiametric nano- and micro-metric morphological characteristics. An example is shown in Figure 3.
The chemical characterization obtained by the EDS spectrometer (Figure 3b) allowed us to discover that these particles are composed mainly of titanium, with the variable presence of iron. Analyzing the histological sections from the same sample, it was possible to observe the presence of some little aggregates directly inside the tissues using micro-Raman spectroscopy. The OM image is reported in Figure 4a.
In this case, the analyses were able to verify the presence of the anatase phase in the particles forming the aggregate [28,29,30], one of the polymorphs of titanium dioxide [10]. In fact, the Raman spectrum of this mineral phase shows a very intense band lying at about 145 cm−1 which is ascribed to Eg modes, together with the bands at about 640 and 200 cm−1 [29]. Finally, the two bands detected at about 515 and 400 cm−1 are related to the anatase A1g and B1g modes, respectively. In addition, micro-Raman mapping was acquired to see if other mineral phases were present (Figure 5).
In the analyzed samples, SEM/EDS allowed us to characterize particles with chemical compositions ascribable to asbestos phases, but their dimensional parameters did not allow us to count them as regulated “asbestos” fibers [22]. In some cases, as with the particles reported in Figure 1, weak signals of Mg and Si with an intensity ratio of the peaks similar to serpentine phases were detected and micro-Raman spectroscopy was applied to understand if a silicate was contained inside the fiber bundles, and it excluded the presence of these minerals.

4. Discussion

The results presented in this paper show the importance of coupling different techniques for the certain characterization of particles and fibers, using both a chemical/morphological and spectroscopic characteristics. In fact, the only analysis carried out using SEM/EDS technique did not allow a certain mineralogical assignment of the inorganic phases. In particular, this technique is envisaged by the Law for asbestos phase identification and the original project did not considered application of micro-Raman spectroscopy for particle characterization. Nevertheless, the findings of other particles with uncertain attribution by SEM/EDS results required the application of micro-Raman spectroscopy, especially in the case of calcium particles, where also Si and Mg were detected, to exclude the presence of an asbestos phase contained inside these particles (Figure 1f).
In the first kind of particles reported in this paper (Figure 1), the chemical characterization detected the presence of calcium, but it was impossible to understand if it was a calcium carbonate, because of the presence of the carbon of the polycarbonate filter. So, it might be classified only as a general “calcium rich particle” [31]. The successive analysis carried out applying micro-Raman spectroscopy allowed to better classify these particles and bundles of fibers also under the mineralogical characteristics, ascribing them to aragonite [25,26], a calcium carbonate polymorph—together with calcite and vaterite—chemical formula CaCO3 [9].
The presence of this mineral phase inside the colonic mucosa might be related to food additives or to antiacid pharmaceuticals. In fact, calcium carbonate additive is utilized in the food industry as a colorant and it also possesses other properties (e.g., acidity regulator, anticaking agent, stabilizer) [32]. Its industrial name is E170 and it is obtained via the processing and cleaning of chalk deposits [32]. Its uses are wide and cover a lot of foods (e.g., chewing gums, desserts, chocolate, cheese, salts, condiments, fish and crustacean pastes, fruit juices and nectars, flavored fermented milk products, edible ices, and baby foods) [32]. The overall acceptable daily intake of calcium carbonates for humans should be below 2500 mg/day, even if there is no value indicated by the Agencies, e.g., the European Food Safety Authority (EFSA), who considered that E170 in food contributes only in a small part to the overall calcium dietary exposure [32]. Moreover, aragonite itself has shown to be an efficient drug delivery system, in particular with its rod-shaped morphology, that allows for a large load of anticancer drugs and acts as an efficient delivery system to cancer cells [33], and the research about calcium carbonate properties both by oral and enteric drug deliveries is very active [34]. In particular, in the work of Render et al., 2016 [34], it was reported that one of the subjects of research regards precisely the enteric drug delivery in the colon.
The second mineral identified in this work was a titanium dioxide polymorph, anatase. Also in this case, Ti dioxide has a large utilization in the field of the food industry: in fact, this compound is also applied mainly as a colorant, in particular as a whitening agent [35]. In Europe, it is labelled as E171. Since 2006, the polymorph phase of anatase—rutile—was authorized to replace it in film coatings for foods but it was demonstrated that anatase is the mainly used form of titanium dioxides in analyzed foods (e.g., chewing gums) [35]. In a recent study [36], E171’s effects were evaluated on a colon cancer cell line after determined times and it was demonstrated that there were biological alterations that could not be reverted after the food additive’s removal. Titanium dioxide was classified as possibly carcinogenic to humans (group 2B) by the International Agency for Research on Cancer (IARC), with inadequate evidence in human studies and sufficient evidence in animal studies [37]. In fact, there was only one article on worker cohorts that highlighted an increased risk of lung cancer, whereas some studies on rats showed an increased risk of lung cancer [37].
Concerning titanium dioxide ingestion, only in 2021 the European Food Safety Authority (EFSA) declared that E171 was not considered a safe food additive [38]. Moreover, an increased tumor growth and progression (but not an increase in formation) was demonstrated in a recent study on a transgenic mouse model for CRC [39]. In addition to this evidence, it was demonstrated that titanium dioxide nanoparticles are able to induce cytotoxicity in colon cancer cell lines [40,41]. The source of exposure might be other than foods or environmental air pollution: in fact, titanium dioxides are utilized in different fields, thanks to their properties. These compounds are applied in different processes, e.g., air-cleaning, water disinfection, antitumor activity, and self-sterilizing [42,43]. Moreover, these compounds are used also in sun cream formulations, thanks to their sunscreen properties [44].
The coupled application of SEM/EDS and micro-Raman spectroscopy allowed us not only to detect the chemical composition and the morphology of these particles, but also to better characterize the mineral phase associated with them, distinguishing them unequivocally as “anatase”.
In this project, the goal was the identification and quantification of asbestos minerals inside tissues derived from patients affected by colorectal cancer but interesting results were also obtained by analyzing other inorganic particles, such as the ones reported in this work.
The identified minerals show two different interactions in the colon. In fact, the carbonate mineral “aragonite” has a positive function in drug delivery, whereas titanium dioxide “anatase” has negative effects on colon cells. In future studies, it might be interesting to systematically analyze all the inorganic particles using the same approach presented in this paper to best characterize the mineral phases identified inside the tissues and to understand where they are located inside the biological medium. Moreover, the same methodology may also be applied to the study of emerging contaminants that might be undistinguishable by means of the SEM/EDS technique alone. An example of such phases is the category of microplastics [45]. In fact, for a complete characterization of this category of pollutants, micro-Raman spectroscopy is one of the best techniques allowing a sure characterization in different media [46,47,48,49,50,51,52], giving spectra that are a “fingerprint” of the different polymers. The advantage of analyzing non-prepared samples by applying SEM/EDS and micro-Raman spectroscopy might also be useful to study polymer additives before and after interaction with tissues; some of them are minerals and they possibly play a role in microplastic toxicity [53,54,55,56].

5. Conclusions

It was demonstrated that the coupled application of SEM/EDS and micro-Raman spectroscopy allows, with no sample manipulation, for identifying not only the morphology and the chemical composition but also the vibrational properties of the different particles/fibers observed inside the tissue. This characteristic might be useful in future studies for understanding the toxicities of the different pollutants for which a clear nexus in disease onset is not defined, in particular when the different phases may crystallize under polymorphs or when the EDS elemental analyses do not surely identify the inorganic phase [57,58,59,60,61,62,63]. Until now, asbestos phases inside the colon tissues of the patients involved in this study have not been detected, but the application of the methodology showed in this work will also be useful in the discrimination of the serpentine phases [4,5,19] observed in organic tissues deriving from patients affected by asbestos-related diseases.

Author Contributions

Conceptualization, A.C.; methodology, A.C.; software, A.C.; validation, A.C., C.R. and D.B.; formal analysis, A.C. and L.C.; investigation, A.C.; resources, C.R.; data curation, A.C. and C.R.; writing—original draft preparation, A.C., D.B., C.R., G.G., A.R., M.B. and L.C.; writing—review and editing, A.C., D.B., C.R., G.G., A.R., M.B. and L.C.; visualization, A.C. and L.C.; supervision, M.B. and A.R.; project administration, A.R., M.B. and A.M.; funding acquisition, A.C. All authors have read and agreed to the published version of the manuscript.

Funding

The present work was funded by the CRT Foundation.

Institutional Review Board Statement

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the Azienda Ospedaliera SS. Antonio e Biagio e Cesare Arrigo of Alessandria (protocol no. 0016369 on 29th July 2020).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

Data are contained within the article.

Acknowledgments

The authors wish to thank Marco Francesco Amisano, Elisabetta Nada, Stefania Erra, and Federica Grosso for their support during sample acquisition, protocol writing, and analysis checks.

Conflicts of Interest

The authors declare no conflicts of interest.

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Minerals 14 00633 g001
Figure 1. (a–e) Elongated particles, with a marked tendency to split into small fibrils along the elongation axis, observed in the three tissue types after digestion. (f) An example of an EDS spectrum recorded from these particles. Na and Cl derive from NaCl crystallization during the digestion procedure.
Figure 1. (a–e) Elongated particles, with a marked tendency to split into small fibrils along the elongation axis, observed in the three tissue types after digestion. (f) An example of an EDS spectrum recorded from these particles. Na and Cl derive from NaCl crystallization during the digestion procedure.
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Minerals 14 00633 g002
Figure 2. (a) Spectrum obtained from the particles as those reported in Figure 1. The bands indicated in red are ascribed to the carbonate mineral phase “aragonite”; the bands in black are ascribed to polycarbonate, the material of which the filter is made where the particles are deposited. In the insert in (a), a particular of the 800–600 cm−1 spectral range, where the symmetric stretching vibrational modes of the Si-Ob-Si bonds of the serpentine phases lie, is reported: it is clear the absence of this mineral phase (highlighted by the red circle). (b) Spectrum obtained on another particle, showing more prominent bands ascribed to “aragonite”.
Figure 2. (a) Spectrum obtained from the particles as those reported in Figure 1. The bands indicated in red are ascribed to the carbonate mineral phase “aragonite”; the bands in black are ascribed to polycarbonate, the material of which the filter is made where the particles are deposited. In the insert in (a), a particular of the 800–600 cm−1 spectral range, where the symmetric stretching vibrational modes of the Si-Ob-Si bonds of the serpentine phases lie, is reported: it is clear the absence of this mineral phase (highlighted by the red circle). (b) Spectrum obtained on another particle, showing more prominent bands ascribed to “aragonite”.
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Figure 3. (a) Aggregate of different isodiametric particles. (b) EDS representative spectrum obtained on the aggregate reported in (a). Na and Cl derive from NaCl crystallization during the digestion procedure.
Figure 3. (a) Aggregate of different isodiametric particles. (b) EDS representative spectrum obtained on the aggregate reported in (a). Na and Cl derive from NaCl crystallization during the digestion procedure.
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Figure 4. (a) Aggregate of different isodiametric particles (indicated by the arrows) observed under micro-Raman spectroscope (magnification 80×). (b) Raman representative spectrum obtained on the particles forming the aggregate reported in (a).
Figure 4. (a) Aggregate of different isodiametric particles (indicated by the arrows) observed under micro-Raman spectroscope (magnification 80×). (b) Raman representative spectrum obtained on the particles forming the aggregate reported in (a).
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Figure 5. (a) OM image reported in Figure 4a: the yellow box highlights the area analyzed with Raman mapping. (b) Example of one Raman spectrum, where anatase was detected. In this spectrum, the bands considered to create the maps are highlighted: anatase one (in blue), paraffin and tissue one (in green), and silicate symmetric stretching of asbestos (in red). (c) Raman map obtained considering the bands indicated in (b). In this map, blue band of anatase reported in (b) is colored in white to best distinguish the particles from the false blue color of the image background.
Figure 5. (a) OM image reported in Figure 4a: the yellow box highlights the area analyzed with Raman mapping. (b) Example of one Raman spectrum, where anatase was detected. In this spectrum, the bands considered to create the maps are highlighted: anatase one (in blue), paraffin and tissue one (in green), and silicate symmetric stretching of asbestos (in red). (c) Raman map obtained considering the bands indicated in (b). In this map, blue band of anatase reported in (b) is colored in white to best distinguish the particles from the false blue color of the image background.
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MDPI and ACS Style

Croce, A.; Bellis, D.; Rinaudo, C.; Cagna, L.; Gatti, G.; Roveta, A.; Bertolotti, M.; Maconi, A. Application of a Multi-Technique Approach to the Identification of Mineral Polymorphs in Histological Samples: A Case of Combined Use of SEM/EDS and Micro-Raman Spectroscopy. Minerals 2024, 14, 633. https://doi.org/10.3390/min14070633

AMA Style

Croce A, Bellis D, Rinaudo C, Cagna L, Gatti G, Roveta A, Bertolotti M, Maconi A. Application of a Multi-Technique Approach to the Identification of Mineral Polymorphs in Histological Samples: A Case of Combined Use of SEM/EDS and Micro-Raman Spectroscopy. Minerals. 2024; 14(7):633. https://doi.org/10.3390/min14070633

Chicago/Turabian Style

Croce, Alessandro, Donata Bellis, Caterina Rinaudo, Laura Cagna, Giorgio Gatti, Annalisa Roveta, Marinella Bertolotti, and Antonio Maconi. 2024. "Application of a Multi-Technique Approach to the Identification of Mineral Polymorphs in Histological Samples: A Case of Combined Use of SEM/EDS and Micro-Raman Spectroscopy" Minerals 14, no. 7: 633. https://doi.org/10.3390/min14070633

APA Style

Croce, A., Bellis, D., Rinaudo, C., Cagna, L., Gatti, G., Roveta, A., Bertolotti, M., & Maconi, A. (2024). Application of a Multi-Technique Approach to the Identification of Mineral Polymorphs in Histological Samples: A Case of Combined Use of SEM/EDS and Micro-Raman Spectroscopy. Minerals, 14(7), 633. https://doi.org/10.3390/min14070633

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