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Non-Contact Surface Morphology Analysis of CO 2 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
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  Non-Contact Surface Morphology Analysis of CO 2  Laser-Irradiated Teethby 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 CO 2  laser irradiation. The surface roughness (  Ra ) was measuredand the results were correlated with a stylus profilometer. Raman analysis was done and the laser irradiation effect after acid etching was alsoexamined. Human intact extracted teeth were vertically sectioned. The laser was applied perpendicularly to non-etched and 35% phosphoricacid-etched teeth. The enamel resulted in a crater-like surface. The material was removed in the places where hydroxyapatite crystallites ranparallel 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 profilesand different image contrast.  Ra  levels obtained by the SEM and CLSM were similar to that obtained with the stylus profilometer. Ramananalysis showed that bands of organic collagen matrix on dentin were lost and broad peaks due to carbon were observed. Both the SEM andCLSM provided non-contact evaluation of structural changes of teeth by laser irradiation through surface analysis in selected microareas, whichwas 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, CO 2  laser  1. Introduction The irradiation effect of the CO 2  laser on teeth has beenstudied from the aspect of surface morphology. CO 2  laserirradiation of the enamel and dentin surface was reported tocause surface fusion. 1) The characterization of the enamelsurface changes according to the wavelength dependence, asrevealed by surface melting, crystal fusion and exfoliation. 2) Watari analyzed the effect of CO 2  laser irradiation for thesame spot on human teeth by employing three differentmicroscopes, a scanning electron microscope (SEM), con-focal laser scanning microscope (CLSM) and atomic forcemicroscope. 3) However, the quantitative analysis of surfacemorphology has been limited. A conventional stylus profil-ometer has usually been used to measure roughness, but themeasurements are separate from SEM imaging. The surfaceroughness (  Ra ) value measured by a single device does notnecessarily offer a comprehensive picture of the surface. 4) Itwas reported that the contact stylus profilometer gives alower  Ra  than an optical profilometer due to the limitations of the spatial dimensions of the stylus in detecting micro-grooves. 5) Furthermore, the stylus profilometer can causedamage to the dental hard tissue due to the contact with thespecimen.The newly developed three-dimensional (3D) analyzingSEM and CLSM can provide imaging and non-contact profileanalyses simultaneously in a selected microarea with highspatial resolution. The CLSM has the advantage of being anon-destructive method since sample preparation is notnecessary, and it also allows subsurface analysis. 6) SEM hashigher resolution and analysis without coating is alsopossible 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 developedusing reflection electrons with 4-quadrant divided detectorsto make possible quantitative surface analysis.Chemical analysis of the laser-irradiated surface is alsoimportant. From this aspect, Raman spectroscopy can giveinformation about the chemical state without damage. 7) Another advantage is that the spatial resolution of about 1micrometer ( m m ) allows chemical and structural informationto be obtained within the  m m  range.This study simultaneously combined imaging and non-profile analyses of surface morphology using the SEM andCLSM, and added compositional information obtained byRaman spectroscopy to evaluate the effects of the CO 2  laseron the enamel and dentin surfaces. The data obtained usingthe SEM and CLSM from the same area of the specimen werealso correlated with the conventional stylus profilometer.Further microscopic observation was done to evaluate theadditional effect of acid-etching pretreatment before laserirradiation by analyzing the results for non-etched and acid-etched dentin. 2. Materials and Methods2.1 Specimen preparation Five freshly extracted, intact, caries-free human thirdmolars and pre-molars were used in this study. They werecleaned and stored in deionized water at 4  C until used.Vertical sectioning parallel to the long axis of each tooth wasdone using a low-speed diamond saw (Minitom, Struers,Copenhagen, Denmark) under running water. Two slabs fromeach tooth with thicknesses of 2mm were obtained. Theexposed enamel and dentin of both slabs were polished withalumina powder (2 m m , 1 m m  and 0.05 m m  successively),rinsed and ultrasonicated in deionized water to remove thepolished debris. One slab of each tooth was kept wet in * Graduate Student, Hokkaido University  Materials Transactions , Vol. 45, No. 4 (2004) pp. 1033 to 1040Special Issue on Frontiers of Smart Biomaterials # 2004 The Japan Institute of Metals  E XPRESS R  EGULAR  A  RTICLE  deionized water until the application of the laser. The otherwas immersed in 35% phosphoric acid for 5 minutes toexpose the collagen fibrils prior to the laser treatment. 2.2 Laser irradiation The laser apparatus used was a CO 2  laser (Model PanalasC10, Panasonic, Osaka, Japan) with a wavelength of 10.6 m m .It was set with a pulse duration time of 0.5s with a 2mmdiameter tip and work distance of approximately 1mm. Asingle pulse irradiation with 3–8W of power was doneperpendicularly to enamel and dentin. 2.3 Observation and surface analysis2.3.1 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 (S-4700 and S-4000, Hitachi, Tokyo, Japan). A 3D analyzer (RD-500W)installed in the SEM S-4700 (3D-SEM) was used for 3D anddepth profile analysis. The S-4700 operates using a cold fieldemission electron source and has high resolution at lowacceleration voltage. The device provides secondary electron(SE) and backscattered electron (BSE) images. The speci-mens were observed at an acceleration voltage of 3kV and nosputter coating was done to allow the same specimen to beexamined by other microscopes. Specimens treated withphosphoric acid were carbon coated and observed using theSEM S-4000 under acceleration voltages of 5kV and 10kV. 2.3.3 Confocal laser scanning microscope (CLSM) The same specimen examined using the SEM S-4700 wasobserved by CLSM (LSM 410 Invert, Carl Zeiss, Oberko-chen, Germany) in the reflection mode through a 543nm He-Ne laser, with an objective lens of   50   and a numericalaperture of 0.85. A series of optical sections parallel to thetooth surface was obtained from each specimen. The pinholewas set to 10, which gave a full width at half maximum(FWHM) peak of 1.33 m m . The distance between sectionswas 0.5 m m . All data were documented at a resolution of  768  576  pixels. The surface topography software allowssurface roughness analysis of the tooth as well as 3Dreconstruction of the images from section series. The surfaceprofile and roughness analyzed using the SEM and CLSMwere then compared. 2.3.4 Stylus profilometer The surface roughness was analyzed using a conventionalstylus profilometer (Surfcom 200C, Tokyo Seimitsu, Tokyo,Japan). The vertical magnification was adjusted to 500K,chart speed 3mm/s and tracing speed 0.03mm/s. The  Ra values were determined as the average height deviation, in m m , from the mean plane through widths of 800 m m  and250 m m  for both enamel and dentin. 2.3.5 Raman spectroscopy Raman spectra of non-irradiated and laser-irradiatedsurfaces were obtained using a Raman spectrometer (Lab-ram, Horiba, Tokyo, Japan). Specimens were excited with aHe-Ne laser (632.8nm) at 40mW through a microscopeobjective lens ( 100  , NA = 0.90). The focal spot size wasabout 1 m m  in diameter. The spectra were recorded at aresolution of 2cm  1 . 3. Results Figure 1 shows a digital microscope image of a verticallysectioned tooth irradiated by the CO 2  laser. Therein, a, b andc indicate that the enamel and dentin were irradiated at 3W,6W and 8W, respectively. The size of lased-area increasedwith the power. Enamel became white and dentin black dueto carbonization of collagen.Figure 2 shows the SEM images and respective depthprofiles of enamel and dentin irradiated at 6W. The surfacesirradiated at 6W were the most representative, and weretherefore chosen for the analysis. The profiles were obtainedin a non-contact mode along the horizontal line in the SEMmicrographs. Enamel showed a crater-like surface, whiledentin had a very smooth surface. Both had cracks due to thethermal shock. The laser-irradiated area became light whitecompared to the surrounding non-irradiated area. The  Ra  was5.12 m m  for enamel and 1.09 m m  for dentin. The square areasdelimited in SEM micrographs of enamel and dentin wereenlarged and compared with those of the CLSM in Figs. 3and 4, respectively.Figure 3 shows the SEM and CLSM images and theirrespective profiles from the identical area of laser-irradiatedenamel. The bright contrast observed at the edges of thegrooves in SEM image became dark in CLSM. The depthprofiles obtained in the non-contact mode showed similaroutlines. The formation of grooves resulted in  Ra  of 3.17 m m for SEM and 6.17 m m  for CLSM analysis.Figure 4 shows a comparison of SEM and CLSM imagesand their respective profiles of laser-irradiated dentin. Withthe SEM, dentinal tubules and cracks were observed.However, with the CLSM the interior of dentinal tubuleswas also observed, differentiating them from the cracks. Thedepth-profile analysis from both the SEM and CLSM showedroughness of 0.12 m m  for SEM and 0.78 m m  for CLSM.Conventional stylus profilometer analysis was also done.The profile was detected by a stylus profilometer that passed800 m m  and 250 m m  from nearly the same area as in Fig. 2.The  Ra  values measured along 800 m m  were 3.63 m m  forenamel and 0.39 m m  for dentin. At 250 m m ,  Ra  was 2.35 m m 1mmaabbcc Fig. 1 Vertical section of tooth irradiated by CO 2  laser. a, b and c indicatedin enamel and dentin were irradiated at 3W, 6W and 8W, respectively.1034 M. K. Yamada, M. Uo, S. Ohkawa, T. Akasaka and F. Watari  -25025200 400 600-25025200 400 600 enamel    D  e  p   t   h ,        d    /      µ   m Width, w  /  µ m dentin Fig. 2 SEM images and respective depth profiles obtained from the irradiated area of enamel and dentin at 6W. The profiles wereobtained in a non-contact mode along the horizontal line in the SEM micrographs. The enclosed areas are enlarged and compared withthose from the CLSM in Figs. 3 and 4. 25 µ m SEM CLSM -2502550 100 150 200    D  e  p   t   h ,        d    /      µ   m Width, w  /  µ m Fig. 3 SEM 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. 2.Non-Contact Surface Morphology Analysis of CO 2  Laser-Irradiated Teeth 1035  for enamel and 0.09 m m  for dentin.The  Ra  results obtained with the SEM and CLSM shown inFigs. 3 and 4 and those for the stylus profilometer arecompared 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 factors between the SEM and CLSM. Thevalues obtained with the SEM were generally smaller thanthose for the CLSM, but larger than with the stylusprofilometer. Dentin had a lower  Ra  than enamel.Figure 6 shows a further enlarged SEM image (a), 3Dimage (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 theenamel prism, which is also called the enamel rod. Eachprism is divided into a head and tail and is constituted of alarge 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 65–70  tothe long axis at the tail. In the tail of the enamel prism shownby the dashed circle, the crystallites diverge laterally forminga flame shape and in the center the crystallite ends appear onthe surface. Regions with crystallites non-parallel to theirradiated surface remains, while the adjacent areas with thecrystallites parallel to the surface have flaked off. The depthprofile (c) shows that the size of the areas where material isremoved was about 5 m m , corresponding to the size of theenamel prism. The grooves formed conferred roughness of 0.4 m m . The three-dimensional image (b) can represent thesurface morphology viewed from any desired angle. In thiscase 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 highermagnification (  5000 ). Cracks were observed mostly insidethe peritubulardentin where the contrast was brighter than forintertubular dentin. It was known from the CLSM image(Fig. 4) that dentinal tubules lay below the peritubular dentinby comparison with the SEM image. The surface roughnesswas 0.17 m m .Figure 8shows the Raman spectra of non-irradiated(a) andirradiated (b) dentin. Phosphate vibrations in hydroxyapatiteare shown at   2  (350–500cm  1 ),   4  (550–650cm  1 ),   1 50 100 150 200-30350 100 150 200 SEM CLSM    D  e  p   t   h ,        d    /      µ   m Width, w  /  µ m 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. 0246    R  a   /      µ   m SEM CLSM StylusProfilometer enamel dentin  3         .  1         7         6         .  1         7        2         .   3         5         0         .  1        2         0         .   7         8         0         .   0         9         Fig. 5 Comparison of SEM, CLSM and conventional stylus profilometermeasurement of surface roughness values (  Ra ) on enamel and dentin.1036 M. K. Yamada, M. Uo, S. Ohkawa, T. Akasaka and F. Watari  (900–1000cm  1 ) and   3  (1000–1130cm  1 ). The organiccollagen matrix peaks appear at 1257, 1470, 1661 and2945cm  1 (Fig. 8a). After irradiation, these peaks were lostand broad peaks due to amorphous carbon were observed inthe range of 1346–1580cm  1 (Fig. 8b). The backgroundintensity became higher. Laser-irradiated enamel alsoshowed a higher background intensity than non-irradiatedenamel. However, the change of enamel was smaller than fordentin. The   2 ,   4 ,   1  and   3  bands were observed after CO 2 laser irradiation, although less intensely.To observe the effectof laser-irradiated collagenon dentin,treatment with 35% phosphoric acid was done and the a b -2.502.55 10 15 20 c    D  e  p   t   h ,        d    /      µ   m Width, w  /  µ m -2025 10 15 20 ca b    D  e  p   t   h ,        d    /      µ   m Width, w  /  µ m Fig. 6 Laser-irradiated enamel surface. (a) Enlarged SEM photo-graph, (b)3D image and (c) cross-sectionprofile from the line in (a).The dashed circle in (a) shows the basic structural unit of the enamelprism.Fig. 7 Laser-irradiated dentin surface. (a) Enlarged SEM photo-graph, (b)3D image and (c) cross-sectionprofile from the line in (a).Non-Contact Surface Morphology Analysis of CO 2  Laser-Irradiated Teeth 1037
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