MICROTOMOGRAPHY STUDY OF BAMBOO IMPREGNATED

WITH SILVER NANOPARTICLES (Ag-NPs)

Omar Pandoli1,a

, Raquel Martins1

,Eric C. Romani2, Sidnei Paciornik

3, HaimonDiniz Lopes Alves

3,

Marcos Henrique de Pinho Maurício3, Fatima Ventura Pereira-Meirelles

1,b, Eric Lobo Luz,

Guilherme, B.Neumann4

, Khosrow Ghavami5,c

1DepartmentofChemistry, PontificiaCatholicUniversityof Rio de Janeiro, Rio de Janeiro, Brazil

2DepartmentofPhysic, PontificiaCatholicUniversityof Rio de Janeiro, Rio de Janeiro, Brazil

3Materials Eng. Department, PontificiaCatholicUniversityof Rio de Janeiro, Rio de Janeiro, Brazil

4DepartmentofBiology, Universidade Pontifícia Católica, Rio de Janeiro, Brazil

5Civil Eng. Department, PUC-RIO andFZEA/USP-Pirassununga, São Paulo, Brazil

aomarpandoli@puc-rio.br,

bmeirelle@puc-rio.br,

cghavami@puc-rio.br

ABSTRACT:

Bamboo is an important non-conventional material used in different areas of engineering. A highly

resistant and malleable giant grass, it presents a high stiffness and strength-to-weight ratio superior

to any wood. Bamboo is composed of cellulose, hemicellulose and lignin and the mechanical

properties are attributed to its fibers. One of the main difficulties in the large-scale use of bamboo is

its low durability, mainly due to fungi and insect attacks. It has been shown that silver nanoparticles

(Ag-NPs) can be effective antimicrobial agent. This paper presents the result of an investigation

using Ag-NPs impregnation of bamboo Dedrocalamus giganteus to improve its resistance to the

attack by fungi, which is the main cause of vegetable decomposition. For this purpose, the starch of

culms was removed through several cycles of washing with warm water. Then, a 5x5x5mm sample

was impregnated with a colloidal solution of Ag-NPs through a vacuum system. X-ray

microtomography (µCT) was used to non-destructively analysis the nanoparticle’s 3D distribution

within the bamboo’s biological matrix. µCT is particularly suited in this case, given the large atomic

weight difference between the Ag particles and the bamboo matrix, leading to significant image

contrast. After image reconstruction and processing, followed by 3D rendering, it was shown that

impregnation was successful, with clusters of Ag-NPs distributed within the parenchyma only. The

antimicrobial activity of homemade and commercial Ag-NPs was evaluated against Aspergillus

niger.

Keywords: silver nanoparticle, impregnation, microtomography, bamboo antimicrobial.

INTRODUCTION:

In today's global vision, preserving the environment, research for sustainable energy sources and

decrease the cost of production has been a major challenge for all fields of technology.

Bamboo is a graminea (Poaceae), found in large quantities in Asia and South America [1,2].

Biocomposites are mainly composed of cellulose (55%), hemicellulose (20%) and lignin (25%), the

latter being responsible for its mechanical strength. The fibers are distributed anisotropically on

longitudinal position. It has rapid growth, with the maximum rate of 22 cm/day, given a kind of

giant bamboo reach up to 40 meters high in few months. Therefore, the use of bamboo has been

evaluated as an interesting alternative in lieu of some composites in various processes used in civil

engineering, textile industry, decoration materials and other industrial processes. Besides, it is also

used in soil remediation [2,3].

The implementation of Bamboo as unconventional materials has increased over the years. Due to

their chemical and mechanical properties, bamboo has shown high versatility. The replacement of

steel in civil construction by bamboo was evaluated in studies at PUC-Rio, obtaining satisfactory

results. According to this study, the values of the modulus of elasticity of bamboo found in the

internal parts, median and external are close to steel and concrete. These data linked to the results of

other tests, ensure the viability of this replacement [1,4]. In addition, the cellulose pulp can act as

reinforcement in the concrete to replace the asbestos fibers [5,6].

However, the application of bamboo has been limited due to their durability, which is affected by

microbiological degradation. Into parenchymal tissue of Bamboo’s biological matrix there are

starch granules, which is an energy source for the growth of microorganisms and is therefore prone

to their attacks, especially by fungi [7,8,9,10].

One proposal to inhibit or reduce the degradation of the vegetable matrix is the treatment with

antifungal compositions. One of the advanced researches, in this regard, is the use of silver

nanoparticles, which actually possess the ability to interfere with the metabolism of microorganisms

[11,12,13,14]. The fungal inhibition mechanism by means of Ag+ ion is not exactly established, but

its effect is cited in few articles, in different fields [15,16,17]. Some studies have been done on the

treatment of natural fibers with nanoparticles, yielding also the antifungal protection for the same

[11,13]. Extending the application to this biological matrix, the functionalization of bamboo with

silver nanoparticles also could prove to be effective to protect against microbiological attacks [18,

19]. Ag-NPs filled up the bamboo matrix could inhibit the proliferation of fungi and thus increase

its durability. The Ag-NPs impregnation can take place advantage of the hydrophilic surface

properties of bamboo fibers [20]. To enhance the dispersion of the Ag-NPs the right choice of

surfactant ligand of Ag-NPs is critical for their loading in to bamboo matrix.

The microtomography (μCT) imaging technique was choice to localize the metal deposition into a

bamboo matrix. Microtomography is a non-destructive technique that relies on the interaction and

attenuation of the radiation when passing through a sample. The technological advancements on X-

ray tubes with smaller focal spot sizes and flat panel detectors made resolution of the micron scale

possible, which, in turn, urged its spread throughout many small to medium laboratories. The result

of μCT is a grayscale image of the internal composition of the sample, which allows for 3D analysis

of the same parameters that were usually limited to a 2D evaluation and provides information on

their distribution. Considering that μCT offers thousands of images, the statistics are far superior to

any other available non-destructive technique. The possibilities of study have been worldly

recognized from several areas of expertise such as orthodontics, biology, earth sciences,

archaeology, and many others [21-30].

In this work, synthesis of Ag-NPs was carried out with microreactor technology, [31, 32] computed

microtomography technique was used to verify the distribution of Ag-NPs into bamboo fibers, and

biological testes were explored to assess the antifungical action of commercial and homemade Ag-

NPs.

EXPERIMENTAL SECTION

Material and Methods. Silver nitrate (AgNO3, >99.9% pure), sodium borohydride (NaBH4, >99%

pure), and trisodium citrate (Na3Citrate, > 99% pure), were purchased from Sigma Aldrich (Brazil).

Double deionized (DI) water with a measured resistivity of 18.2 mΩ cm-1

was used to make all the

solutions for the desired reactions. For the synthesis of silver nanoparticle, in flow mode, were used

two syringe pumps purchased from Future Chemistry and one glass micro reactor from Micronit.

The syringe pumps are able to inject the solutions of silver precursor and organic ligand into the

micro channel device to improve the mixing and the formation of the organic-metal complex in a

real short time compare with the batch mode. PFA (perfluoroalkoxyalkane) tubes and PEEK

(polyetheretherketone) connections, purchased from UpChurch, were used to inject the two

solutions into the microreactor with 6 μL internal volume. The bamboo impregnation experiments

were carried out using a 4 years old Dendrocalamus giganteus bamboo culm obtained from

FZEA/USPPirassununga, SP.Commercial silver colloids Ag-NPs, with diameters of 20, 40 and 60

nm purchased from TED –Pella Inc, were supplied in water with a negative surface charge of

sodium citrate. Microbiological tests were performed with the fungus Aspergillus niger strains,

grown in liquid Sabouroud, Kasvi supplied by Slabor.

Synthesis of silver nanoparticles. Silver nanoparticles (Ag-NPs) were synthesized employing

NaBH4 as reducing agent, and the organic ligand, Na3Citrate, as stabilizer. At first step of organic-

metal complex formation, Na3Citrate organic ligand with three carboxylate functional groupies able

to complex silver ion (Ag+) into the microchannel devise. At the second step, during the chemical

reduction of Ag+ to silver metal nanoparticle, Na3Citrate acts as capping agents to prevent the

aggregation of Ag-NPs. The reduction process was conducted in continuous flow reaction, where

AgNO3 solution (10-2

and 10-3

mol L-1

) was mixed with sodium citrate organic ligand (10-2

and 10-

3mol L

-1) into a glass microreactor system. The two solutions were injected by means of two syringe

pumps at flow rate of 0,25mL min-1

. The Ag+: ligand complex flowing out from the micro reactor

dropped directly into the fresh NaBH4 solution(10-2

and 10-3

mol L-1

) under vigorous stirring at room

temperature. The home made silver colloids (NP-01 at 10-2

mol L-1

and NP-02 at 10-3

mol L-1

) were

characterized with UV-VIS spectrophotometer (Perkin-Elmer 950 lambda) and Scanning

Transmission Electron Microscopy (STEM). The colloidal solutions NP-01 and NP-02 were used

for the anti microbial tests, meanwhile only the higher concentrated solution NP-01 was used for

impregnation test of a bamboo specimen.

Morphological characterization (STEM).For morphological characterization of silver

nanoparticles using microscopy, a small drop (2.5 μL) of nanoparticles solution (NP-02) was placed

on a carbon transmission electron microscopy grid (holey carbon) and allowed to evaporate

completely in air before analysis. A field emission scanning electron microscope (FEG-SEM)

(JEOL, JSM-6701F) was operated in the transmission mode (STEM) at 30 kV with a work distance

of 6.0 mm using the bright-filed detector.

Microbiological Test. The antimicrobial activity of Ag-NPs was evaluated. For this purpose,

Aspergillus niger was cultivated in Sabouroud liquid medium at 29oC and 160 rpm in the presence

and absence of Ag-NPs. Medium without Ag-NPs and/or cells were used as negative and positive

controls, respectively. Moreover, a commercial colloidal solution of Ag-NPs (CO) with diameters of

20 nm, 40 nm and 60 nm was also used for comparison. The growth of the fungi was performed in

15 ml tubes and 50 ml erlenmyer, being accompanied by 24 and 48 hours. After the growth 48hs,

the systems (yeast + medium) were vacuum filtered, dried in oven at 60oC and weighed on

analytical balance accuracy.

Bamboo impregnation. A cubic section of bamboo with the follow dimensions, 5x5x5 mm, was

placed into test tube with 0,50mL of colloidal solution of Ag-NPs (NP-01). It was submitted to five

impregnation cycles through a vacuum system. After each cycle a new fresh NP-02 was used. Soon

after, the impregnated bamboo with Ag-NPs was analysed in with X-ray microtomograph.

X-Ray Microtomography (MicroCT).It was used a Zeiss X radia Versa 510 microtomograph for

the X-ray microtomography experiment. This system consists of a X-ray microfocus source, up to

160 kV voltage and 10 W power and a detector where a two-stage magnification technique is

employed: i. geometric magnification as with conventional µCT, ii. a scintillator converts X-rays to

visible light, which is then optically magnified. Achievable true spatial resolution up to 0.7 µm.

The bamboo sample was scanned with the following setup:

Voltage (kv) 80

Power (w) 7

Pixel size (µm) 4

Exposure Time (s) 0.2

Number of Projections 1600

RESULTS AND DISCUSSION

Surface plasmons resonance (SPR) band of a diluted silver colloidal solution is presented in figure 1.

According with UV-VIS spectra, the maximum wavelength (λmax) of the SPR band is centred at

390.7 nm and 394 nm respectively to NP-01 and NP-02. This λmax is characteristic for nanoparticles

with size diameter between 10-20 nm. The size nanoparticle was estimated by the empirical

equation (1) with a value of 11.46 nm and 16,19 nm, respectively. The relation between the

diameter of the silver nanoparticles and the absorption maximum is given by the following relation,

in which D is the particle diameter in nm and λmax

is the absorption maximum of the nanoparticles in

nm:

D = - 0,005441λ2max + 5,654λmax – 1367 (1)

300 350 400 450 500 550 600 650 700

0,0

0,2

0,4

0,6

0,8

1,0

1,2

Ab

so

rbâ

ncia

(nm)

NP-01

NP-02

CO-20

CO-40

CO-60

Figure 1. UV-VIS spectra of diluted colloidal solution of homemade Citrate capped Ag-NPs with

λmax of the SPR band centered at 390.7 nm and 394 to NP-01 and NP-02, respectively.

On the same graph, one can see the spectra of commercial nanoparticles of 20 nm, 40 nm and 60 nm,

which have absorption at the following wavelengths: 398 nm, 409 nm and 430 nm. For the reading

in the spectrophotometer, all nanoparticles were diluted in the ratio 1: 4, with the exception of the

NP-01, was diluted in the ratio 1:40.

The validity of calculated empirical diameter value of Ag-NPs was confirmed by STEM analysis.

Figure 2 shows a typical STEM image of the silver nanoparticles synthesized by the reduction of

AgNO3 in the presence of sodium citrate and NaBH4 (NP-01). In order to extract statistical

distribution of silver nanoparticles, image analysis (IA) method using a routine created in the image

J program was applied. For image analysis, intensity threshold, segmentation and post-processing

methods were used in order to detect the individual silver nanoparticles [33]. Using this procedure,

it was possible to measure the major axis, minor axis among others morphological attributes of

silver nanoparticles. The inset of figure 2 shows the histogram on the statistical distribution of the

diameter of the silver nanoparticles with a mean value of 14.3 ± 3.6 nm obtained using the image

analysis procedure. This histogram was constructed from the average of the series statistics on

major and minor axes of the silver nanoparticles.

Figure 2. STEM image of silver nanoparticles with Na3Citrate as organic stabilizer. Inset: size

diameter histogram for Ag-NPs (NP-01) used for the bamboo impregnation.

X-Ray Microtomography analysis is presented in figures 3 and 4, where 3D images show bamboo

samples, respectively, before and after silver colloids impregnation. In figure 3, the internal section

of pure bamboo is showed. The light gray colour shows the lignificated vegetal cells of the

sclerenchyma tissue around the bundle vessel. The dark gray colour represents the parenchyma

structure of the bamboo matrix.

Figure3.MicroCTimages of bamboo specimen before Ag-NPs colloidal solution treatment.

In figure 4, 3D images with different contrast colours allow to identify silver aggregates inside

bamboo sample. In this case: red colour represents the bamboo matrix, while yellow colour

represents silver aggregates deposited on it. Although the X-Ray Microtomography analysis didn’t

show nanoparticles deposition, due to the limitation of the spatial resolution of this technique,

different perspectives of the specimen help us to identify in which regions silver aggregates are

penetrated and concentrated. Silver aggregates with dimensions between 10-20 μm were found

concentrated preferentially into parenchyma structure. This can be explained because the

parenchyma structure is the tissue in which the vegetal cell are still alive, where the biopolymer

lignin, whom supports the vegetal cells, is opened and let go in and out water, mineral salt and other

materials useful to sustain life cell.

Figure 4. MicroCT 3D images of bamboo after five cycles impregnation of Ag-NPs. Red and yellow

colours represent, respectively, bamboo matrix and silver aggregates.

Figure 5 presents the results obtained from antimicrobial test, in tests on tubes. It can be seen that

cell dry weight obtained after 48 of cultivation on the tube containing NP-01 was superior to the

control. While in the tubes NP-02 and CO-20, we observe the greater growth inhibition.

Figure 5. Dry-weight of fungi culture in the absence or presence of Ag-NPs. CO is the commercial

colloidal solution of Ag-NPs with different diameters (20,40 and 60nm).

Many factors can interfere with the development of microorganisms, such as temperature, oxygen

availability and amount of nutrients. However, the development of concurrent experiment with the

control, assures us that the change in the mycelium growth is related to the unique modified

variable: the presence and the nature of the nanoparticles. Thus, it is understood that the mycelium

weight decreased or increased by the presence of the Ag-NPs. In this sense, NP-02 showed greater

efficiency in inhibiting the mycelium growth compared not only to the NP-01, but also in relation to

the commercial Ag-NPs. When we compare CO-20, CO-40 and CO-60, it is also noticed that as

greater is the size of NP as lower is the inhibition effect.

As NP-02 was the most effective in the previous test, it was used as follows, in the experiments in

shaken-flasks (Table 2). Table 2 shows the results obtained after 48 and 72h of cultivation. The

images confirm the results obtained in the experiment conducted in the tube where the NP-02 lead

to the lower growth.

Table 2 - Micelium aggregation images of Aspergillus niger grown in the absence (Control) or in

presence of Ag-NPs (NP-02) and commercial Ag-NPs (CO-20) in 50 mL erlenmeyer flasks

(a)

Control NP-02 CO-20

48 h

72 h

CONCLUSIONS

In our best knowledge this is the first report in which X-Ray Microtomography analysis is used to

study a biocomposite metal-biological matrix, where is possible identify a penetration and the

deposition of metal silver into bamboo parenchyma structure. Further studies are focused on the

radial distribution of Ag-NPs into bamboo fibers, as well as the variation of impregnation as a

function of the number of impregnation cycles. Further research will evaluate the effect of different

electric charged surfactants for the Ag-NPs on their dispersion, and the impact on bamboo

degradation. Possible previous chemical treatments of bamboo fibers can directly interfere with

adhesion of the nanoparticles and improve the impregnation of the Ag-NPs.

From the antimicrobial tests, NP-02 was more effective to inhibit mycelium formation. Due to

different inhibition degree obtained with NP-02 and CO-20, it can be suggested that they act in a

different way over the fungi culture. It is well known the effect of NP-Ag in bacteria, prokaryotic

cells, but there are few works with fungi, eukaryotic cells. It´s worth mentioning that bamboo is also

eukaryotic. Thus, it is important to thoroughly investigate the interaction between Ag-NPs and

fungi, and understand it, in order to use Ag-NPs as protector of bamboo biological matrix against

fungi.

Finally, our results suggest that homemade silver colloidal solution with NP-02 can be used in the

further impregnation experiments and assess the bamboo durability decreasing fungi proliferation

into biological vegetal matrix.

REFERÊNCES

1. K. Ghavami, Bamboo as reinforcement in structural concrete elements, Cement & Concrete

Composites. 27 (2005) 637–649.

2. H. P. S. A. Khaliul, I.U. H. Bhat, M. Jawaid, A. Zaidon, D. Hermawan, Y. S. Hadi, Review

Bamboo fiber reinforced biocomposites. Materialdesing: 42 (2012) 353-368.

3. N. P. Marinho, S. Nisgoski, U. Klock, A. S.Andrade, G. I. B. Muñiz, Análise Química Do

Bambu-Gigante (Dendrocalamus giganteus Wall. ex Munro) Em Diferentes Idades. Ciência

Florestal, Santa Maria, v. 22, n. 2, p. 413-418, abr.-jun., 2012.

4. D. E. Hebel, A. Javadian, F. Heisel, K. Schlesier, D. Griebel, M. Wielopolski, Process-

controled optimization of the tensile strength of bambu fiber composite for strutural aplication.

Composites: Parte B 67 (2014) 125-131.

5. V. C. Correa, S. F. Santos, G. Marmol, A. A. S. Curvelo, H. Savastano Jr, Potential of

bamboo organosolv pulp as a reinforcing element in fiber–cement materials. Construction and

Building Materials 72 (2014) 65-71.

6. P. Zakikhani, R. Zahari, M. T. H. Sultan, D. L. Majid, Extration and preparation of bamboo

fibre-reinforced composites. Materials and desing 63 (2014) 820-828.

7. A. Azzini, Amido a partir do bambu. Bragantia, Campinas, 43(1):45-50, 1984.

8. A. Azzini, A. L. Beraldo, Determinação de fibras celulósicas e amido em cavacos laminados

de três espécies de bambu gigante. SCIENTIA FORESTALIS n. 57, p. 45-51, jun. 2000.

9. K. D. Hyde, D. Zhou, T. Dalisay, Bambusicolous fungi: A review. Fungal Diversity 9: 1-14,

2002.

10. E. Tanaka, Mechanisms of bamboo witches’ broom symptom development caused by

endophytic/epiphytic fungi. Plant Signaling & Behavior 5:4, 415-418; April 2010.

11. F. Z. Hang, X. Wu, Y. Chen, A. H. Lin, Application Of Silver Nanoparticles To Cotton

Fabric As An Antibacterial Textile Finish, Fibbers And Polymers. 10 (2009) 496-501.

12. J. A. Dahl, B. L. S. Maddux, j. E. Hutchison, Toward Greener Nanosynthesis. Chem. Rev.

2007,107,2228−2269.

13. M. D. Teli, J. Sheikh, Study Of Grafted Silver Nanoparticle Containing Durable

Antibacterial Bamboo Rayon, Cellulose Chem. Technol. 47 (2013) 69-75.

14. Z. M. Xiu, Q. B. Zhang, H. L. Puppala, V. L. Colvin, P. J. J. Alvarez, Negligible Particle-

Specific Antibacterial Activity Of Silver Nanoparticles, Nano Lett. 12 (2012) 4271−4275.

15. A. F. Wady, Propriedade antifungica de uma resina para base de prótese modificada com

nanopartículas de prata. Dissetação. Universidade Estadual Paulista. Araraquara, SP. 2011.

16. K. S. Woo, K. S. Kim, K. Lamsal, Y. J. Kim, S. B. Kim, M. Jung, S. J. Sim, H. S. Kim, S.J.

Chang, J. K. Kim, Y. S. Lee, An In Vitro Study Of The Antifungal Effect Of Silver Nanoparticles

On Oak Wilt Pathogen Raffaelea Sp., J. Microbiol. Biotechnol. 19 (2009) 760–764.

17. S. W. Kim, J. H. Jung, K. Lamsal, Y. S. Kim, J. S. Min, Y. S. Lee, Antifungal Effects Of

Silver Nanoparticles Against Various Plant Pathogenic Fungi, Microbiology.40(2012)53-58.

18. M. D. Teli, J. Sheikh, Modified Bamboo Rayon-copper nanoparticle composites as

antibacterial textiles. International journal of biological macromolecules 61 (2013) 302-307.

19. B. Tang, L. Sun, J. Li, J. Kaur, H. Zhu, S. Qin, Y. Yao, W. Chen, X. Wang, Funcionalization

of Bamboo Pulp Fabrics with Noble Metal Nanoparticles. Dyes and Pigments (2013) 289-298.

20. C. A. Fuentes, L. Q. N. Tran, M. V. Hellemont, V. Janssens, C. Dupoint-Gillain, A. W. V.

Vuure, I. Verpoest, Effect of physical adhesion on mechanical behaviour of bamboo fiber reinforced

thermoplastic composites. Colloids and Surfaces A: Physicochem. Eng. Aspects 418 (2013) 7-15.

21. R. A. Ketcham, W. D. Carlson, Acquisition, optimization and interpretation of X-ray

computed tomographic imagery: applications to the geosciences. Computers & Geosciences 27

(2001) 381–400.

22. I. Lima, C. Appoloni, L. Oliveira, R. T. Lopes, Caracterização De Materiais Cerâmicos

Através Da Microtomografia Computadorizada 3d. Revista Brasileira de Arqueometria, Restauração

e Conservação. Vol.1, No.2, pp. 022 - 027.

23. J. S. Fernandes, C. R. Appoloni, C. P. Fernandes, Determinação de Parâmetros

Microestruturais e Reconstrução de Imagens 3-D de Rochas Reservatório por Microtomografia de

Raios X. Revista Ciências Exatas e Naturais, Vol.11 nº 1, Jan/Jun 2009.

24. H. Alves, I. Lima, J. T. Assis, M. Geraldes, R. T. Lopes. Comparison of pore space features

by thin sections and X-ray microtomography. Appl. Radiat. Isot. 2014, 94(12), 182–190.

25. G. De-Deus, J. Marins, A. d. A. Neves, C. Reis, S. Fidel, M. A. Versiani, H. Alves, R. T.

Lopes, S. Paciornik. Assessing accumulated hard-tissue debris using micro-computed tomography

and free software for image processing and analysis. J. Endod. 2014, 40(2), 271–276.

26. G. De-Deus, E. J. N. L. Silva, J. Marins, E. Souza, A. d. A. Neves, F. G. Belladonna, H.

Alves, R. T. Lopes, M. A. Versiani. Lack of causal relationship between dentinal microcracks and

root canal preparation with reciprocation systems. J. Endod. 2014, 40(9), 1447–1450.

27. A. A. Neves, E. Coutinho, M. V. Cardoso, S. V. Jaecques, B. V. Meerbeek. Micro-CT based

quantitative evaluation of caries excavation. Dent. Mater. 2010, 579–588.

28. L. Haili, R. Swennen, A. Foubert, M. Dierick, P. Jacobs. 3D quantification of mineral

components and porosity distribution in Westphalian C sandstone by microfocus X-ray computed

tomography. Sediment. Geol. 2009, 220(1–2), 116–125.

29. R. A. Ketcham, W. D. Carlson. Acquisition, optimization and interpretation of X-ray

computed tomographic imagery: applications to the geosciences. Comput. Geosci. 2001, 27, 381–

400.

30. V. Cnudde, M. N. Boone. High-resolution X-ray computed tomography in geosciences: a

review of the current technology and applications. Earth-Sci. Rev. 2013, 123, 1–17.

31. X. Dong, X. Ji, H. Wu, L. Zhao, J. Li, W. Yang, Shape Control of Silver Nanoparticles by

Stepwise Citrate Reduction. J. Phys. Chem. C 2009, 113, 6573–6576.

32. A. H. L. Machado, O. Pandoli, L. S. M. Miranda, R. O. M. A. de Souza, Micro Reatores:

Novas Oportunidades em Síntese Química, Revista Virtual Quim. 6 (2014) 1076-1085.

33. S. Paciornik and M. H. P. Mauricio, “Digital imaging” in ASM Handbook Vol. 9

Metallography and Microstructures, G. F. V. Voort (ASM International, Materials Park, OH, 2004).