Open access peer-reviewed chapter

Laser-Induced Breakdown Spectroscopy and Microscopy Study of Human Dental Tissues

Written By

Muhammad Mustafa, Anwar Latif and Majid Jehangir

Submitted: 10 April 2022 Reviewed: 25 April 2022 Published: 24 May 2022

DOI: 10.5772/intechopen.105054

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Electron Microscopy

Edited by Mohsen Mhadhbi

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Abstract

Laser-induced breakdown spectroscopy (LIBS) analysis of human dental tissues: enamel and dentine, performed by utilizing Nd: YAG laser (𝜆=1064 𝑛𝑚, 𝜏=6 𝑛𝑠, 𝐸=50 𝑚𝐽) to investigate threshold ablation of laser energy density. Quantitative results based on the experiment provide us with threshold ablation value of laser energy density for calcium (Ca) ablation in enamel and dentine tissues. The computed threshold laser energy density for Ca ablation in dentin tissue is 0.38 J/cm2, which is significantly lower than the threshold in the enamel, which is 1.41 J/cm2. Scanning electron microscopic (SEM) examination of dental tissues determines that the dentin surface contains pores, voids, and bubbles that make it easy to ablate at low laser energy density, while enamel has a closely packed smear layer structure that is difficult to ablate, requiring high energy densities. These findings are helpful in the field of laser dentistry, where lasers are widely used for dental treatment.

Keywords

  • electron microscopy
  • laser-induced breakdown spectroscopy
  • Nd: YAG laser
  • dental tissues
  • energy density

1. Introduction

Lasers have been utilized as a supplementary treatment in dentistry since 1964. They have experienced significant advancements in a variety of dental applications. Nd: YAG lasers are mainly used in dentistry, including soft and hard tissue surgery, cavity preparation in tooth enamel and dentine, detection of tooth decay, prevention of tooth decay by modifying the crystalline structure of enamel, and tooth whitening [1, 2, 3, 4, 5].

Lasers to ablate tissues may cause cracks, fractures, fissures, irradiation roughness, surface irregularities, and the removal of smeared layers [6]. The risks of the laser can be avoided by selecting optimized laser parameters according to the chemical composition of the irradiated tissues [7, 8]. It is significant to determine the minimum value of laser energy density that is required to take out an atom of material of the same element, known as threshold ablation. To optimize the laser energy density and energy of the laser, it is required to calculate the threshold ablation of energy density for calcium (the most commonly found mineral in dental tissues) in enamel and dentine that can be precisely achieved by laser-induced breakdown spectroscopy [9, 10].

LIBS is an atomic emission spectroscopic technique. It is also known as “laser-induced plasma spectroscopy” (LIPS) because elements and atomic species are quantified through spectrometric analysis of laser-induced plasmas. The LIBS spectroscopy technique uses a high-intensity laser capable of ablating small amounts of material, thereby creating a short-duration plasma [11]. The formed plasma contains the excited atoms, molecules, and ions that appeared in the target. As the plasma cools, the atoms, ions, and molecules lose energy due to the emission of light photons that carry certain wavelengths [12]. Thus, spectroscopic characterization of the plasma light will reveal the elements present in the target. The identification of many elemental lines, including both the wavelength and the intensity within the emission spectrum, will form a unique spectral fingerprint of the target, such as calcium [13, 14, 15, 16].

Scanning electron microscopy (SEM) is a versatile technique to investigate surface topography, structural morphology, composition, the orientation of grains, crystallography, etc. of dental tissues by achieving its three-dimensional (3D) image with high quality and spatial resolution [17]. In SEM, micro to nanostructure analysis can be examined by focusing a finely collimated electron beam on the dental slices. Due to interaction between incident electrons and the slices, various types of signals may emit from irradiated tissues such as secondary electrons (SE), backscattered electrons (BSE), Auger electrons, continuous and characteristics X-rays, and other photons of different energies [18]. These signals are detected by suitable detectors available in SEM such as Everhart–Thornley detector (ETD) is used to detect SE [19], BSE using a retractable circular BSE detector (CBS) [20], and X-rays signal by energy dispersive spectrometer (EDS) detector [21]. 3D highly magnifying image form on the computer by connecting the signal detectors through the optical fiber.

In the present research work, we have calculated the threshold ablation value of laser energy density for calcium found in human dental tissues such as enamel and dentine by employing LIBS. To investigate the significant difference in threshold ablation for Ca in dental tissues SEM analyses are conducted to examine the surface and structural morphology of these tissues.

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2. Sample preparations and methods

Two identical samples of sound extracted human molar teeth were collected from a Dental clinic, Medical & Dental Curatives, and Implant Center, approved by the institute of public health, the government of Punjab, Pakistan. The molars were immersed in saline solution to avoid dryness. Teeth were sectioned into their longitudinal axis using a diamond disk (model HOR, Horico, Germany) to produce two subsamples of dental tissues enamel and dentin of each 5 × 5 × 2 mm thickness. For experimental purposes, enamel-dentine surfaces were polished using #600 and #1200 silicon carbide sandpapers.

Sample 2 slices were coated with an electrically conductive layer of copper for SEM observation.

Figure 1 displays a schematic diagram of the LIBS experimental setup used to record the LIBS spectra of dental tissues in a vacuum. For experimentation J200 Tandem LA (Applied Spectra, Inc. USA) LIBS unit is used, in which pre-installed Q-switched Nd: YAG (1064 𝑛𝑚, 6 𝑛𝑠, 50 𝑚𝐽) Quanta Ray Pro-230-10 Spectra physics, USA, with the flattop beam profile, the spot diameter of 0.09 cm, and energy density range of 1–15 J/cm2 was projected as the source of irradiation. The sample chamber in J200 is a box equipped with an adjustable sample stage. The slices of sample 1: enamel and dentine, are sequentially placed in the sample chamber and irradiated by the Nd: YAG laser. An infrared (IR) lens of a focal length of 7.5 cm focuses the laser beam on slices to generate plasma. The emission of light photons from the plasma is collected by a lens adjusted at an angle of about 45° to the laser beam axis and fed to the spectrometer. The Scanning Czerny-Turner type spectrometers are continuously operating to capture and read data. It has a response range of wavelength (190–900) nm with a spectral resolution of 0.2 nm. At various laser energy densities of 1 J/cm2 to 15 J/cm2, maximum emission intensities of spectra are displayed by connecting spectrometers to a linear charge-coupled device (CCD) camera.

Figure 1.

Represents a schematic diagram of the LIBS experimental setup for dental tissue analysis.

FEI Nova 450 Scanning electron microscope (SEM) was used for micro to nanostructural analysis of dental tissues. Copper-coated sample 2 slices are placed into a 110 x 110 mm/150 x 150 mm stage that can be positioned in five directions: x, y, z, rotation, and tilt. These movements are motorized and controlled by xT microscope control software. The immersion mode is selected from the xT microscope-driven user interface drop-down menu. The immersion lens is activated, and the lens detector (TLD) in secondary electron operation amplifies images of specimens over 500,000×, resulting in ultra-high-resolution imaging in digital format.

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3. Statistical analysis

Axiom laser ablation (LA) is an operating software in laser-induced breakdown spectroscopy. A chemo-metric technique in LA is a statistical mechanism for the identification of elements to quantify them. It also integrates ICP-MS data management and analysis tools, which are essential for generating precise quantitative solutions and highly accurate statistics. It is employed to choose isotopes of interest and compare their temporally resolved ICP-MS outcomes. Time-resolved signal device is used to study the time-resolved behavior of elements displayed on the graph at various time intervals. Time-resolved ICP-MS rapidly smooths the data and TRSD (Temporal Relative Standard Deviation) statistics are easily obtained. Graphical Development Tool (GDT) chemo-metric software from LIBS Spectra allows us to distinguish LIBS spectra and visualize the differences. The calcium lines that appeared in the LIBS spectra were chosen as the ablation indicators. They appeared at a variety of wavelengths. Their peak areas were computed and plotted according to the laser energy densities. The relationship between peak areas and energy densities was then represented via data fitting. The ablation thresholds were determined based on the curve fitting. The calculations and statistical analysis were performed using Origin (8.5) software. The measurements from SEM micrographs were taken by ImageJ (1.53 k) software.

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4. Results

The relationship between the peak areas of calcium lines and full-width half-maximum is given below [22]:

A=h×FWHM2.35×0.3989E1

Here, A represents the peak area, h is the amplitude of the peak, and FWHM is the full-width half-maximum. Eq. (1) provides a simple way of peak area calculation by measuring peak height and a full-width half-maximum of spectra. Areas of peak to corresponding energy densities are used to calculate the threshold ablation of calcium in enamel and dentin. In this study, Ca peaks are the peaks of interest that directly reflect the concentration of Ca within the dental tissues.

In the J200 LIBS unit the Nd: YAG laser (1064 nm, 6 nm, 50 mJ) focused on tooth sample enamel and dentin that formed plasma. The emission of photons from plasma is captured by the spectrometers and then displayed on computer intensity spectra as a function of wavelength, as shown in Figures 2 and 3 respectively. The area under the curves was calculated using the built-in Axiom laser ablation software for laser-induced breakdown spectroscopy.

Figure 2.

Spectra obtained at different energy densities for enamel at the 14th shot.

Figure 3.

Spectra at different energy densities for dentin at the 4th shot.

4.1 Enamel spectra obtained by J200 tandem LA-LIBS instrument using axiom LA system software at various laser energy densities

Figure 2 represents spectra of enamel tissue for different energy density ranges 3.7 J/cm2 to 14.8 J/cm2 and optimized number of pulses (14). The emission of the discrete line from enamel tissue is identified as a calcium element, and they have three main features: wavelength, intensity, and shape. The calcium element in tissues has different energy levels and the transition between these energy levels determines the wavelengths of emitted spectral lines.

Table 1 displays the data for 11 enamel spectra (in Figure 2) at different wavelengths and their corresponding peak areas. The maximum peak area was 32 counts/nm at 14.8 J/cm2 while a minimum of 5 counts/nm at 3.7 J/cm2 and 4.25 J/cm2 was attained.

Energy Density (J/cm2)Integrated Peak Areas at different Intensities (Count per nm)
At 395.5 nm, 397.5 nmAt 409.5 nmAt 416.5 nm, 417.5 nm, 419.3 nmAt 423.2 nm, 426.4 nmAt 431.2 nm
3.786.56.855.6
4.2586.56.9556
5.8109.78.467.3
6.81110.896.58
7.851111.1106.710
8.81112.110711
10.1141214912.8
11.22119161216
12.42320201419.1
13.62726231720
14.832292819.4825

Table 1.

Peak area calculation for calcium in enamel using axiom LA software.

4.2 Dentine spectra obtained by J200 tandem LA-LIBS instrument using axiom LA system software at various laser energy densities

Figure 3 exhibits the spectra of dentine tissue for the laser energy densities 2.4 J/cm2 to 14.8 J/cm2 and the optimized number of pulses 4. Ca metals were identified as the discrete lines emitted from dentine tissue, and they had three primary characteristics: wavelength, intensity, and shape. Ca element in tissues has different energy levels, which determine the wavelength of lines.

Table 2 illustrates the results of 12 dentine spectra (of Figure 3) at various wavelengths and their associated peak areas. The intensity (count/nm) of the integrated peak area increases as energy density increases. The largest peak area was 26.4 counts/nm at 13.6 J/cm2, and a minimum of 3 counts/nm at laser energy densities of 2.4 J/cm2, 3.7 J/cm2, and 4.25 J/cm2.

Energy Densities (J/cm2)Integrated Peak Areas at different Intensities (Counts/nm)
At 395.5 nm, 397.5 nmAt 409.5 nmAt 416.5 nm, 417.5 nm, 419.3 nmAt 423 nm, 426.4 nmAt 431.2 nm
2.44.33433.2
3.74.844.233.6
4.256.85.3634
5.865.86.23.14.3
6.88.77.66.13.15.1
7.8513.611.47.83.46.1
8.816.113.18.73.47
10.116.2138.83.46.6
11.21713.28.13.86.2
12.4242010.44.28
13.626.420.212.44.69.6
14.82419.813.24.89.8

Table 2.

Peak area calculation for calcium in dentine using axiom LA software.

4.3 Surface morphological analysis of enamel and dentin

Figure 4 represents electron micrographs of dental tissues obtained by SEM at 1000× magnification. The enamel surface is not very smooth, covered with smear layers, and a few tiny holes throughout the surface are shown in Figure 4(a). Pores, bubbles, and debris are examined in dentin in Figure 4(b). The measured mean area of particle debris is 2.1 μm2 and the mean distance between two consecutive debris is 0.6 μm in dentin tissue. In both tissues, smeared Layers observed which formation of micro and nanocrystalline structures is, bit blurriness is an obstacle act of layers in the field of vision and imaging.

Figure 4.

SEM micrographs of sample2 right after cutting with diamond disc (a) enamel slice (b) dentin slice.

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5. Discussion

In the present research, we employed two spectroscopic techniques (LIBS & SEM), which were complemented by electron microscopy. When a high-power pulsed laser beam hits the target, it causes localized heating and vaporization of the sample materials. The ablated material expands and forms a plasma plume. Hence, there is a relationship between plasma intensity and ablated material. LIBS is used to define an element in a sample and plasma intensity. Emission intensity is linearly correlated to the number of elements in the sample [10].

Figure 5 represents the plot of peak area versus energy density (there are five exponential lines: purple, green, blue, black and red at intensities of (395.5 nm, 397.5 nm), (409.5 nm), (416.5 nm, 417.5 nm, 419.3 nm), (423.2 nm, 426.4 nm) and 431.2 nm, respectively. There are 11 points in each curve that represent peak areas at different energy densities. The line of best fit is drawn which intercepts the x-axis at 1.41 J/cm2 and gives the threshold ablation value of energy density for enamel at the 14th shot.

Calcium emission wavelength (nm)Equationsslopex-intercept
395.5397.5y=4.4655e0.1287x2.11.418
409.5y=3.9591e0.133x1.91.415
416.5, 417.5, 419.3y=3.8412e0.1295x1.81.413
423.2, 426.4y=3.3446e0.1358x1.21.410
431.2y=2.7536e0.1287x1.71.414

Line of best fit: slope: 2.10 x-intercept: 1.41.

Threshold ablation for calcium in enamel: 1.41 J/cm2.

Figure 5.

Threshold energy density of ablation for calcium in the enamel.

The integrated peak area for calcium at (395.5 nm, 397.5 nm), (409.5 nm), (416.5 nm, 417.5 nm, 419.3 nm), (423.2 nm, 426.4 nm), and 431.2 nm were plotted for different energy densities in Figure 6. There are five linear lines: purple, green, blue, black and red. Each line has 12 points representing peak areas at different energy densities. The line of best fit is drawn such that the intercept x-axis at 0.38 J/cm2 gives the threshold ablation value of energy density for dentine at the 4th laser pulse (shots).

Calcium emission wavelength (nm)Equationsslopex-intercept
395.5397.5y=1.64x0.6281.60.38
409.5y=1.46x0.5641.40.38
416.5, 417.5, 419.3y=1.05x0.4181.00.39
423.2, 426.4y=0.58x0.2220.50.38
431.2y=0.70x0.2770.70.38

Line of best fit: slope: 1.64 X-intercept: 0.38.

Threshold ablation for calcium in enamel: 0.38 J/cm2.

Figure 6.

Threshold energy density of ablation for calcium in dentine.

It can be spotted from Figures 5 and 6 that the peak areas of enamel and dentine Ca lines are set up at laser energy densities of 3.35 J/cm2 and 2.43 J/cm2 respectively. By data fitting curves analysis, an exponential fit is observed for enamel and a linear fit for dentine. The curves in (Figures 5 and 6), combine to intersect the x-axis that gives us threshold ablation, in enamel and dentine tissues respectively. Outcomes of LIBS revealed that the threshold ablation for Ca in enamel is approximately four times that of the threshold ablation for Ca in dentine. SEM analysis was conducted to figure out the reason for the huge difference in threshold ablations for calcium in dental tissues by examining their surface topography and structural properties. SEM micrographs in Figure 4 show that enamel has smear layers that are close to each other’s making its structure rigid, hard and calcified. Pores, bubbles, and open spaces on the dentine surface make it delicate, flexible, and less calcified, which is a primary reason for having lower threshold ablation for Ca as compared to enamel [23, 24, 25, 26, 27].

Chemical compositional studies of dental tissues determine that they contain hydroxyapatite crystal (HAP), the main mineral constituent of teeth, which is the most stable and least soluble, form of calcium phosphate. The average size of HAP is larger in enamel than dentine, which makes the former more calcified. In addition, enamel contains 95% inorganic material, 1% organic material, and 4% water by weight percentage, whereas dentin is composed of 70% inorganic material, 2% organic material, and 10% water by weight percentage [28, 29, 30]. Hence, the compositional analysis revealed that enamel has a lower concentration of water and higher mineral content (than dentin) which makes it difficult to ablate.

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6. Conclusions

The LIBS technique has been successfully applied to compute threshold ablation for calcium in dental tissues. Findings revealed that enamel has a higher threshold of ablation for calcium than dentine. SEM microstructural observations and measurements suggest that enamel has a closely packed structure of smear layers making it hard whereas a high porosity level in dentin causes less toughness. These findings are beneficial in the field of dentistry for the use of lasers in dental treatment.

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Acknowledgments

We are grateful to Dental Habitat and Dental & Curatives and Implant Center for providing experimental facilities to conduct our research work.

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Conflict of interest

The authors declare no conflict of interest.

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Appendices and nomenclature

LASER

light amplification by stimulated emission and radiation

Nd

YAG, Neodymium-doped yttrium aluminum garnet

LIBS

laser-induced breakdown spectroscopy

LA

laser ablation

LIPS

laser-induced plasma spectroscopy

IR

infrared

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Written By

Muhammad Mustafa, Anwar Latif and Majid Jehangir

Submitted: 10 April 2022 Reviewed: 25 April 2022 Published: 24 May 2022