**4. Results**

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

$$A = \frac{h \times FWHM}{2.35 \times 0.3989} \tag{1}$$

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

*Laser-Induced Breakdown Spectroscopy and Microscopy Study of Human Dental Tissues DOI: http://dx.doi.org/10.5772/intechopen.105054*

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 laserinduced breakdown spectroscopy.

## **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.

## **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

**Figure 3.** *Spectra at different energy densities for dentin at the 4th shot.*

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


#### **Table 1.**

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


#### **Table 2.**

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

*Laser-Induced Breakdown Spectroscopy and Microscopy Study of Human Dental Tissues DOI: http://dx.doi.org/10.5772/intechopen.105054*

**Figure 4.** *SEM micrographs of sample2 right after cutting with diamond disc (a) enamel slice (b) dentin slice.*

peak area increases as energy density increases. The largest peak area was 26.4 counts/nm at 13.6 J/cm<sup>2</sup> , and a minimum of 3 counts/nm at laser energy densities of 2.4 J/cm<sup>2</sup> , 3.7 J/cm<sup>2</sup> , and 4.25 J/cm<sup>2</sup> .

#### **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 μm<sup>2</sup> 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.

### **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.


*.*

*Threshold ablation for calcium in enamel: 1.41 J/cm<sup>2</sup>*

#### **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).


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

*Threshold ablation for calcium in enamel: 0.38 J/cm<sup>2</sup> .*

#### *Laser-Induced Breakdown Spectroscopy and Microscopy Study of Human Dental Tissues DOI: http://dx.doi.org/10.5772/intechopen.105054*

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/cm<sup>2</sup> and 2.43 J/cm<sup>2</sup> 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 [23–27].

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 [28–30]. 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.

**Figure 6.** *Threshold energy density of ablation for calcium in dentine.*
