Non Contact Surface Morphology Analysis of CO2 Laser Irradiated Teeth by Scanning Electron Microscope and Confocal Laser Scanning Microscope

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Non-Contact Surface Morphology Analysis of CO

2

Laser-Irradiated Teeth

by Scanning Electron Microscope and Confocal Laser Scanning Microscope

Magda Kiyoko Yamada

*

, Motohiro Uo, Shoji Ohkawa, Tsukasa Akasaka and Fumio Watari

Division of Dental Materials and Engineering, Department of Oral Health Science, Graduate School of Dental Medicine, Hokkaido University, Sapporo 060-8586, Japan

A newly developed scanning electron microscope (SEM) installed with a 3D analyzer and a confocal laser scanning microscope (CLSM) were used to evaluate the images and surface profiles of enamel and dentin after CO2laser irradiation. The surface roughness (Ra) was measured

and the results were correlated with a stylus profilometer. Raman analysis was done and the laser irradiation effect after acid etching was also examined. Human intact extracted teeth were vertically sectioned. The laser was applied perpendicularly to non-etched and 35% phosphoric acid-etched teeth. The enamel resulted in a crater-like surface. The material was removed in the places where hydroxyapatite crystallites ran parallel to the irradiated surface. Non-etched dentin showed little change after irradiation, with some cracks mostly in the peritubular dentin; whereas in acid-etched dentin there was evaporation of collagen and melting of hydroxyapatite. The SEM and CLSM showed similar profiles and different image contrast.Ralevels obtained by the SEM and CLSM were similar to that obtained with the stylus profilometer. Raman analysis showed that bands of organic collagen matrix on dentin were lost and broad peaks due to carbon were observed. Both the SEM and CLSM provided non-contact evaluation of structural changes of teeth by laser irradiation through surface analysis in selected microareas, which was not possible using the stylus profilometer. Dentin showed more structural changes on the acid-etched surface by irradiation than on the non-etched one.

(Received November 7, 2003; Accepted February 2, 2004)

Keywords: scanning electron microscope (SEM), confocal laser scanning microscope (CLSM), tooth, acid etching, CO2laser

1. Introduction

The irradiation effect of the CO2 laser on teeth has been

studied from the aspect of surface morphology. CO2 laser

irradiation of the enamel and dentin surface was reported to

cause surface fusion.1) The characterization of the enamel

surface changes according to the wavelength dependence, as

revealed by surface melting, crystal fusion and exfoliation.2)

Watari analyzed the effect of CO2 laser irradiation for the

same spot on human teeth by employing three different microscopes, a scanning electron microscope (SEM), con-focal laser scanning microscope (CLSM) and atomic force

microscope.3)However, the quantitative analysis of surface

morphology has been limited. A conventional stylus profil-ometer has usually been used to measure roughness, but the measurements are separate from SEM imaging. The surface

roughness (Ra) value measured by a single device does not

necessarily offer a comprehensive picture of the surface.4)It

was reported that the contact stylus profilometer gives a

lowerRathan an optical profilometer due to the limitations of

the spatial dimensions of the stylus in detecting

micro-grooves.5) Furthermore, the stylus profilometer can cause

damage to the dental hard tissue due to the contact with the specimen.

The newly developed three-dimensional (3D) analyzing SEM and CLSM can provide imaging and non-contact profile analyses simultaneously in a selected microarea with high spatial resolution. The CLSM has the advantage of being a non-destructive method since sample preparation is not

necessary, and it also allows subsurface analysis.6)SEM has

higher resolution and analysis without coating is also possible if a low acceleration voltage is used. Until now, however, only the imaging results have been observed.

Recently, a SEM with a 3D-analyzer has been developed using reflection electrons with 4-quadrant divided detectors to make possible quantitative surface analysis.

Chemical analysis of the laser-irradiated surface is also important. From this aspect, Raman spectroscopy can give

information about the chemical state without damage.7)

Another advantage is that the spatial resolution of about 1

micrometer (mm) allows chemical and structural information

to be obtained within themmrange.

This study simultaneously combined imaging and non-profile analyses of surface morphology using the SEM and CLSM, and added compositional information obtained by

Raman spectroscopy to evaluate the effects of the CO2laser

on the enamel and dentin surfaces. The data obtained using the SEM and CLSM from the same area of the specimen were also correlated with the conventional stylus profilometer. Further microscopic observation was done to evaluate the additional effect of acid-etching pretreatment before laser irradiation by analyzing the results for non-etched and acid-etched dentin.

2. Materials and Methods

2.1 Specimen preparation

Five freshly extracted, intact, caries-free human third molars and pre-molars were used in this study. They were

cleaned and stored in deionized water at 4C until used.

Vertical sectioning parallel to the long axis of each tooth was done using a low-speed diamond saw (Minitom, Struers, Copenhagen, Denmark) under running water. Two slabs from each tooth with thicknesses of 2 mm were obtained. The exposed enamel and dentin of both slabs were polished with

alumina powder (2mm, 1mm and 0.05mm successively),

rinsed and ultrasonicated in deionized water to remove the polished debris. One slab of each tooth was kept wet in

*Graduate Student, Hokkaido University

Special Issue on Frontiers of Smart Biomaterials

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deionized water until the application of the laser. The other was immersed in 35% phosphoric acid for 5 minutes to expose the collagen fibrils prior to the laser treatment.

2.2 Laser irradiation

The laser apparatus used was a CO2laser (Model Panalas

C10, Panasonic, Osaka, Japan) with a wavelength of 10.6mm.

It was set with a pulse duration time of 0.5 s with a 2 mm diameter tip and work distance of approximately 1 mm. A single pulse irradiation with 3–8 W of power was done perpendicularly to enamel and dentin.

2.3 Observation and surface analysis 2.3.1 Digital microscope

Digital Microscope (VH-6300, Keyence Corporation, Osaka, Japan) was used and the specimen was observed at

25 magnification.

2.3.2 SEM

The specimens were analyzed using SEMs (4700 and S-4000, Hitachi, Tokyo, Japan). A 3D analyzer (RD-500W) installed in the SEM S-4700 (3D-SEM) was used for 3D and depth profile analysis. The S-4700 operates using a cold field emission electron source and has high resolution at low acceleration voltage. The device provides secondary electron (SE) and backscattered electron (BSE) images. The speci-mens were observed at an acceleration voltage of 3 kV and no sputter coating was done to allow the same specimen to be examined by other microscopes. Specimens treated with phosphoric acid were carbon coated and observed using the SEM S-4000 under acceleration voltages of 5 kV and 10 kV. 2.3.3 Confocal laser scanning microscope (CLSM)

The same specimen examined using the SEM S-4700 was observed by CLSM (LSM 410 Invert, Carl Zeiss, Oberko-chen, Germany) in the reflection mode through a 543 nm

He-Ne laser, with an objective lens of 50 and a numerical

aperture of 0.85. A series of optical sections parallel to the tooth surface was obtained from each specimen. The pinhole was set to 10, which gave a full width at half maximum

(FWHM) peak of 1.33mm. The distance between sections

was 0.5mm. All data were documented at a resolution of

768576 pixels. The surface topography software allows

surface roughness analysis of the tooth as well as 3D reconstruction of the images from section series. The surface profile and roughness analyzed using the SEM and CLSM were then compared.

2.3.4 Stylus profilometer

The surface roughness was analyzed using a conventional stylus profilometer (Surfcom 200C, Tokyo Seimitsu, Tokyo, Japan). The vertical magnification was adjusted to 500 K,

chart speed 3 mm/s and tracing speed 0.03 mm/s. The Ra

values were determined as the average height deviation, in

mm, from the mean plane through widths of 800mm and

250mmfor both enamel and dentin.

2.3.5 Raman spectroscopy

Raman spectra of non-irradiated and laser-irradiated surfaces were obtained using a Raman spectrometer (Lab-ram, Horiba, Tokyo, Japan). Specimens were excited with a He-Ne laser (632.8 nm) at 40 mW through a microscope

objective lens (100, NA = 0.90). The focal spot size was

about 1mm in diameter. The spectra were recorded at a

resolution of 2 cm 1.

3. Results

Figure 1 shows a digital microscope image of a vertically

sectioned tooth irradiated by the CO2laser. Therein, a, b and

c indicate that the enamel and dentin were irradiated at 3 W, 6 W and 8 W, respectively. The size of lased-area increased with the power. Enamel became white and dentin black due to carbonization of collagen.

Figure 2 shows the SEM images and respective depth profiles of enamel and dentin irradiated at 6 W. The surfaces irradiated at 6 W were the most representative, and were therefore chosen for the analysis. The profiles were obtained in a non-contact mode along the horizontal line in the SEM micrographs. Enamel showed a crater-like surface, while dentin had a very smooth surface. Both had cracks due to the thermal shock. The laser-irradiated area became light white

compared to the surrounding non-irradiated area. TheRawas

5.12mmfor enamel and 1.09mmfor dentin. The square areas

delimited in SEM micrographs of enamel and dentin were enlarged and compared with those of the CLSM in Figs. 3 and 4, respectively.

Figure 3 shows the SEM and CLSM images and their respective profiles from the identical area of laser-irradiated enamel. The bright contrast observed at the edges of the grooves in SEM image became dark in CLSM. The depth profiles obtained in the non-contact mode showed similar

outlines. The formation of grooves resulted inRaof 3.17mm

for SEM and 6.17mmfor CLSM analysis.

Figure 4 shows a comparison of SEM and CLSM images and their respective profiles of laser-irradiated dentin. With the SEM, dentinal tubules and cracks were observed. However, with the CLSM the interior of dentinal tubules was also observed, differentiating them from the cracks. The depth-profile analysis from both the SEM and CLSM showed

roughness of 0.12mmfor SEM and 0.78mmfor CLSM.

Conventional stylus profilometer analysis was also done. The profile was detected by a stylus profilometer that passed

800mmand 250mmfrom nearly the same area as in Fig. 2.

The Ra values measured along 800mm were 3.63mm for

enamel and 0.39mmfor dentin. At 250mm,Rawas 2.35mm

1mm a

a

b

b c

c

Fig. 1 Vertical section of tooth irradiated by CO2laser. a, b and c indicated

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-25 0 25

200 400 600

-25 0 25

200 400 600

enamel

Depth,

d

/

µ

m

Width, w/µm

dentin

Fig. 2 SEM images and respective depth profiles obtained from the irradiated area of enamel and dentin at 6 W. The profiles were obtained in a non-contact mode along the horizontal line in the SEM micrographs. The enclosed areas are enlarged and compared with those from the CLSM in Figs. 3 and 4.

25

µ

m

SEM

CLSM

-25 0 25

50 100 150 200

Depth,

d

/

µ

m

Width, w/µm

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for enamel and 0.09mmfor dentin.

TheRaresults obtained with the SEM and CLSM shown in

Figs. 3 and 4 and those for the stylus profilometer are

compared in Fig. 5. The figure shows that the order of Ra

values was the same and that there were differences of 1.95-to 6.5-fold in fac1.95-tors between the SEM and CLSM. The values obtained with the SEM were generally smaller than those for the CLSM, but larger than with the stylus

profilometer. Dentin had a lowerRathan enamel.

Figure 6 shows a further enlarged SEM image (a), 3D image (b) and depth profile (c) of laser-irradiated enamel.

The depth profiles were obtained from the line shown in (a). The dashed circle (a) shows the basic structural unit of the enamel prism, which is also called the enamel rod. Each prism is divided into a head and tail and is constituted of a large numbers of hydroxyapatite crystallites. These crystal-lites run parallel along the long axis of the prism at the head,

and diverge gradually from this to become angled 65–70to

the long axis at the tail. In the tail of the enamel prism shown by the dashed circle, the crystallites diverge laterally forming a flame shape and in the center the crystallite ends appear on the surface. Regions with crystallites non-parallel to the irradiated surface remains, while the adjacent areas with the crystallites parallel to the surface have flaked off. The depth profile (c) shows that the size of the areas where material is

removed was about 5mm, corresponding to the size of the

enamel prism. The grooves formed conferred roughness of

0.4mm. The three-dimensional image (b) can represent the

surface morphology viewed from any desired angle. In this case it is the upper left.

Figure 7 shows the SEM image of Fig. 2(a), 3D image (b) and depth profile (c) of laser-irradiated dentin at higher

magnification (5000). Cracks were observed mostly inside

the peritubular dentin where the contrast was brighter than for intertubular dentin. It was known from the CLSM image (Fig. 4) that dentinal tubules lay below the peritubular dentin by comparison with the SEM image. The surface roughness

was 0.17mm.

Figure 8 shows the Raman spectra of non-irradiated (a) and irradiated (b) dentin. Phosphate vibrations in hydroxyapatite

are shown at 2 (350–500 cm 1), 4 (550–650 cm 1), 1

50 100 150 200

-3 0 3

50 100 150 200

SEM

CLSM

Depth,

d

/

µ

m

Width, w/µm

Fig. 4 SEM and CLSM images and their respective profiles of laser-irradiated dentin obtained from the same area of the same specimen. The images are enlargements of the areas delimited in Fig. 2.

0 2 4 6

Ra/

µ

m

SEM

CLSM

Stylus

Profilometer

enamel dentin

3.17

6.17

2.35

0.12

0.78

0.09

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(900–1000 cm 1) and 3 (1000–1130 cm 1). The organic

collagen matrix peaks appear at 1257, 1470, 1661 and

2945 cm 1 (Fig. 8a). After irradiation, these peaks were lost

and broad peaks due to amorphous carbon were observed in

the range of 1346–1580 cm 1 (Fig. 8b). The background

intensity became higher. Laser-irradiated enamel also

showed a higher background intensity than non-irradiated enamel. However, the change of enamel was smaller than for

dentin. The2,4,1and3bands were observed after CO2

laser irradiation, although less intensely.

To observe the effect of laser-irradiated collagen on dentin, treatment with 35% phosphoric acid was done and the

a

b

-2.5 0 2.5

5 10 15 20

c

Depth,

d

/

µ

m

Width, w/µm

-2 0 2

5 10 15 20

c

a

b

Depth,

d

/

µ

m

Width, w/µm

Fig. 6 Laser-irradiated enamel surface. (a) Enlarged SEM photo-graph, (b) 3D image and (c) cross-section profile from the line in (a). The dashed circle in (a) shows the basic structural unit of the enamel prism.

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exposed collagen fibrils were irradiated with the laser. Figure 9 shows SEM images of non-etched (a) and acid-etched (b) dentin after laser irradiation. Non-acid-etched lased dentin changed to a slightly white contrast in the SEM image. The morphology of irradiated and non-irradiated areas was not much different (a). On the other hand, the lining of dentinal tubules on acid-etched laser-irradiated dentin looked more markedly white (b). Outside of the irradiated area, a band of melted material was observed (arrows).

Figure 10 shows SEM micrographs of non-etched and acid-etched dentin before (a) and after (b) laser irradiation at higher magnification. Non-etched dentin did not show much change in structure after laser irradiation (b). Cracks were observed on some parts of peritubular dentin. After acid etching, the exposed collagen fibrils were apparent on dentin (a). These collagen fibrils disappeared with application of the

laser and melted hydroxyapatite was observed (b).

4. Discussion

4.1 Imaging and depth-profile analysis of teeth irradi-ated by the CO2laser

The SEM (BSE) and CLSM had different contrast levels. Groove-surface images such as those observed on enamel after laser irradiation gave rise to strong contrast with the CLSM, emphasizing the irregular surface (Fig. 3). The inside under the surface could also be viewed with limited depth as

observed for dentinal tubules in Fig. 4. Correlating the Ra

results for the SEM and CLSM, it was observed that the CLSM tended to produce larger values. This may be explained by the difference between the scanning methods. The image of the SEM is formed with the numbers of electrons reflected from the surface while the CLSM obtained the images from a series of optically sliced sections parallel

to the tooth surface. The lower resolution of about 0.1mm

limited by the light wavelength and the optically sectioned

slice thickness reasonably set to 0.5mm(Z-interval) in CLSM

may limit the mechanical scanning. Although there are other methods to improve the value of the resolution, a resolution

of around 0.5mmmay limit the detection of roughness less

than that.

The conventional, contact stylus profilometer was

em-ployed using the same scanning line 250mm in length as

obtained from SEM and CLSM for comparison. However, the selection of the same area was approximate and precise selection was difficult. Using the stylus profilometer, the

results for Ra did not provide much detail of the surface

compared to the SEM and CLSM. The Rawas the lowest,

probably due to the limitation of the stylus profilometer tip. The CLSM provides subsurface examination of intact

tooth samples,3) which have transparency to some extent,

offering additional data to those obtained by conventional

0 1000 2000 3000 2

ν ν ν ν1

3 4

a

b

Intensit

y (a.u.)

Wave number,

κ

/cm

-1

Fig. 8 Raman spectra of dentin before (a) and after irradiation (b). The1–

4peaks are attributed to phosphate and arrows to collagen.

irradiated

non-irradiated

150

µ

m

irradiated

non-irradiated

a

b

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methods such as the SEM. Although there are advantages regarding the CLSM technique such as the capacity of determine the depth profile analysis from 3D reconstructions of the cavity shape and cross sections without the need for

sample cutting,8) Watson warned against using 3D

recon-struction made from serial optical sections due to the low resolution in the optical axis of the microscope that could

cause deformation in the image.9) On the other hand, the

SEM’s higher resolution results in well-defined detailed images, especially at higher magnifications. SEM 3D images also offer details of tooth surface morphology and roughness after laser irradiation, which makes it possible to construct images observed from any angle.

The conventional stylus profilometer is useful for analysis of large areas, but the selection of desired area on a microscale is not possible. The topographic SEM and CLSM provide quantitative surface analysis from the microarea correspondent to a microscopic image for a delicate surface like the tooth via a non-contact method.

4.2 Raman spectra

The background intensity of Raman spectra on dentin became higher after laser irradiation. It is known that this, mostly contributed by fluorescence, is lower as the binding is

tighter.10) Therefore, the high background intensity in the

laser-irradiated surface suggests breakage of the cross-linking in the structure. Bands attributed to the organic

collagen matrix observed at 1257, 1470, 1661 and 2945 cm 1

disappeared after CO2 laser irradiation and peaks attributed

to amorphous carbon appeared instead in the range of 1346–

1580 cm 1 (Fig. 8b). This finding suggests that laser

irradiation instantly heats the tooth surface to a high

temperature, resulting in the burning and decomposition of collagen and formation of amorphous carbon. Enamel showed small changes compared to dentin. This is due to the differences in morphological structures and physical properties of the dental hard tissues. Enamel is composed mostly of inorganic material (hydroxyapatite) with no collagen. The higher background intensity observed after laser irradiation of enamel showed that the hydroxyapatite structure was partly decomposed.

4.3 Effects of CO2 laser irradiation on enamel and dentin

The absorption of laser light in dental hard tissue is

important for the ablation of teeth11) and is very much

dependent on the wavelength of the laser light.12)Dental hard

tissue has absorption bands ranging from 9.0 to 11.0mm.

Therefore, a CO2 laser with a wavelength of 10.6mm has

strong absorption in enamel and dentin. Although CO2laser

has a high absorption rate by water (Absorption Coefficient,

AC = 103), the absorption of hydroxyapatite is higher,

resulting in an increase in temperature.13) Thus, thermal

shock may cause cracks in the tooth.

Besides the influence of the laser wavelength, power, pulse duration, number of pulses and spot size in the radiation effect, the absorption of laser light also depends on the nature of the biological tissue. Enamel is a highly mineralized tissue composed of 96% inorganic material (hydroxyapatite crys-tallites), 1% organic material and 3% water. Although enamel has a low tensile strength and is brittle, its rigidity and the flexible support of the underlying dentin minimize the possibility of fracture. Dentin is composed by 70% inorganic material, 20% organic material and 10% water. The

non-etched

acid-etched

a

b

10

µ

m

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content of the fibrous protein collagen, the main component of the organic material, is much higher than in enamel. This may explain the carbonization of dentin.

3D-SEM observation of laser-irradiated enamel at high magnification (Fig. 6) showed that the laser had a tendency to detach material in the head of the enamel prism where

crystallites ran parallel to the long axis of the prism. CO2

laser-induced surface alteration depends on the prism orientation, and consequently, on the orientation of the

hydroxyapatite crystallites. Ferreiraet al.also found

flaked-off material to be more common in parallel than in

perpendicular prism orientation to the specimen surface.14)

It was suggested that the cracks formed due to the thermal shock parallel to the irradiated enamel, if allowed to propagate along the lines of weakness between prisms and crystals, would cause flaking off or ejection of material. On the other hand, when crystals are in transverse orientation in the irradiated surface such as those observed in the dashed circle in Fig. 6, the lines of weakness would generally lie perpendicular to the irradiated surface. This would impede the propagation of cracks and hence partially inhibit the ejection of material. On dentin, cracks were induced inside the peritubular dentin (Fig. 7). The content of hydroxyapatite in this area is higher than in intertubular dentin. Thus, the structure is brittle and more susceptible to crack formation by thermal shock.

4.3.1 Effect of acid etching prior to laser irradiation In Fig. 9 the difference of contrast between non-etched and acid-etched dentin after laser irradiation can be seen. The laser-irradiated area became white. This contrast change was stronger on acid-etched dentin. This suggested that the average atomic numbers of inorganic and organic compo-nents were higher in the irradiated area than in non-irradiated, and more marked in acid-treated dentin. This is in accordance with the fact that hydroxyapatite is formed by heavier atoms (Ca, P), not the light atoms (C, N, O, H) found in collagen. In Fig. 9b, there is a band of melted material around the laser-irradiated area (arrows). One possible explanation for the appearance of this structure is that the explosive pressure caused by the instant heat of laser irradiation may have arrested the melted structure from the center, depositing and consolidating the material in the periphery.

Pretreatment using acid etching much increased the effect of laser irradiation on the structural modification of dentin. The collagen fibrils left after acid etching evaporated and melted hydroxyapatite was observed (Fig. 10). There was not much change on the non-etched dentin after laser irradiation. This may be because in non-etched dentin, the collagen and

apatite are mixed. Although the CO2 laser light is highly

absorbed by water, it is much more absorbed by hydrox-yapatite. Therefore, the underlying layer of hydroxyapatite left under the collagen fibrils after acid etching may have a tendency to more directly be affected by the laser. Thus, the addition of acid etching pretreatment enhanced the laser effect.

5. Conclusions

(1) The SEM and CLSM provided non-contact profile

analysis and the Ra of the irradiated tooth surface in

selected microareas in a plane with high resolution, which was an additional advantage over the conven-tional stylus profilometer.

(2) The SEM and CLSM showed inverse contrast and their respective non-contact surface roughness analyses could quantitatively evaluate the surface profile and roughnesses of enamel and dentin after laser irradiation. (3) Raman spectroscopy suggested that the hydroxyapatite structure was partly decomposed and confirmed the carbonization of collagen in dentin after laser irradi-ation.

(4) CO2 laser-induced surface alteration depends on the

crystallite orientation of the enamel prism. Regions with crystallites nonparallel to the irradiated surface remained, whereas areas with crystallites parallel to the surface flaked off.

(5) The laser effect on dentin was enhanced by the addition of acid-etching pretreatment. The acid-etched dentin caused the disappearance of fibril collagen and melting of hydroxyapatite, whereas the non-etched surface did not show much change under microscopic analysis, except for some cracks on peritubular dentin.

Acknowledgments

We would like to thank: Dr. Takaki Kumazawa, Dr. Yoji Kamiura and the staff of Kumazawa Dental Clinic for the use

of the CO2 laser; Prof. Minoru Wakita of Hokkaido

University for discussions relating to enamel structure; Dr. Yoshinobu Nodasaka and Mr. Toshi Sugawara for their kind help, and Hitachi High-Technologies Corporation for their 3D analysis using SEM.

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Figure

Fig. 1Vertical section of tooth irradiated by CO2 laser. a, b and c indicatedin enamel and dentin were irradiated at 3 W, 6 W and 8 W, respectively.
Fig. 1Vertical section of tooth irradiated by CO2 laser. a, b and c indicatedin enamel and dentin were irradiated at 3 W, 6 W and 8 W, respectively. p.2
Fig. 2SEM images and respective depth profiles obtained from the irradiated area of enamel and dentin at 6 W
Fig. 2SEM images and respective depth profiles obtained from the irradiated area of enamel and dentin at 6 W p.3
Fig. 3SEM and CLSM images and their respective profiles of laser-irradiated enamel obtained from the same area of the same specimen.The images are enlargements of the areas delimited in Fig
Fig. 3SEM and CLSM images and their respective profiles of laser-irradiated enamel obtained from the same area of the same specimen.The images are enlargements of the areas delimited in Fig p.3
Fig. 4SEM and CLSM images and their respective profiles of laser-irradiated dentin obtained from the same area of the same specimen.The images are enlargements of the areas delimited in Fig
Fig. 4SEM and CLSM images and their respective profiles of laser-irradiated dentin obtained from the same area of the same specimen.The images are enlargements of the areas delimited in Fig p.4
Fig. 5Comparison of SEM, CLSM and conventional stylus profilometermeasurement of surface roughness values (Ra) on enamel and dentin.
Fig. 5Comparison of SEM, CLSM and conventional stylus profilometermeasurement of surface roughness values (Ra) on enamel and dentin. p.4
Fig. 6Laser-irradiated enamel surface. (a) Enlarged SEM photo-
Fig. 6Laser-irradiated enamel surface. (a) Enlarged SEM photo- p.5
Fig. 7Laser-irradiated dentin surface. (a) Enlarged SEM photo-graph, (b) 3D image and (c) cross-section profile from the line in (a).
Fig. 7Laser-irradiated dentin surface. (a) Enlarged SEM photo-graph, (b) 3D image and (c) cross-section profile from the line in (a). p.5
Fig. 9Comparison of laser irradiation effect of non-etched (a) and acid-etched (b) dentin by SEM
Fig. 9Comparison of laser irradiation effect of non-etched (a) and acid-etched (b) dentin by SEM p.6
Fig. 8Raman spectra of dentin before (a) and after irradiation (b). The �1–�4 peaks are attributed to phosphate and arrows to collagen.
Fig. 8Raman spectra of dentin before (a) and after irradiation (b). The �1–�4 peaks are attributed to phosphate and arrows to collagen. p.6
Fig. 10SEM micrographs of non-etched and acid-etched dentin before (a) and after (b) laser irradiation at high magnification.
Fig. 10SEM micrographs of non-etched and acid-etched dentin before (a) and after (b) laser irradiation at high magnification. p.7

References

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