**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 , 6 , 50 ) 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

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

#### **Figure 1.**

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

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/cm<sup>2</sup> to 15 J/cm2 , maximum emission intensities of spectra are displayed by connecting spectrometers to a linear charge-coupled device (CCD) camera.

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.

## **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.
