Modern Research and Educational Topics in Microscopy. FORMATEX 2007 A. Mndez-Vilas and J. Daz (Eds.

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Examination of dentin surface using AFM and SEM

Roman Kubinek11, Zdenka Zapletalova2, Milan Vujtek1, Radko Novotn3, Hana Kolarova4 and Hana Chmelickova5
Department of Experimental Physics, Faculty of Science, Palack University, Olomouc, Czech Republic 1st Clinic of Stomatology, Medical Faculty, Palack University, Olomouc, Czech Republic 3 Department of Microscopy methods, Medical Faculty, Palack University, Olomouc, Czech Republic 4 Department of Medical Biophysics, Medical Faculty, Palack University, Olomouc, Czech Republic 5 Joint Laboratory of Optics, Palack University and Institute of Physics of AS CR, Olomouc, Czech Republic
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Atomic force microscopy (AFM) as one of the techniques of Scanning Probe Microscopy is useful for imaging of surface structures. This method can yield 3D high-resolution topographic images of sample surfaces by using a scanning technique for conductors and insulators on atomic scale. A special construction of AFM scanner enables to observe biological samples in liquid environments. Artefacts caused by dehydration of samples are removed this way. Dentin of human teeth is a vital hydrated tissue. It is strongly sensitive to dehydration and drying that are commonly used in preparation of samples in examinations by Scanning Electron Microscopy (SEM). We describe our experience in examination of dentin surfaces of extracted human third molars using contact method of AFM under moist conditions.
Keywords: Atomic Force Microscopy (AFM); Scanning Electron Microscopy (SEM); dentin; smear layer

1. Introduction
Scanning Probe Microscopy (SPM) is a set of experimental methods used in imaging of surface structures at subatomic resolution [1]. Besides physics and chemistry of surfaces, the method is useful also in biological sciences. One of the clones of SPM is Atomic Force Microscopy (AFM). AFM is based on mapping of an atomic-force field on a surface of an examined sample. Both conductive and nonconductive samples can be studied this way. According to the type of the contact between the tip of the cantilever and the sample, AFM can operate in three modes: contact mode, noncontact mode an intermittent contact (tapping) mode. In case of non-contact mode the tip vibrates in the direction perpendicular to the surface, the value of its amplitude is in order of tens of nm and resonance frequency is 200 kHz. Because there is no mechanical contact it is possible to measure also soft and elastic samples. Intermittent contact resembles the contact and non-contact mode. This mode is most suitable for biological samples. It is used when damage of the examined sample could be caused by friction or drawing. AFM provides a real topographical three-dimensional image of a sample surface with vertical resolution from 0.1 nm and lateral resolution from 0.1 nm. The obtained resolution depends on the given sample. It gives a computer image in real time and so can be used for monitoring of dynamic processes. The greatest advantage of AFM applications in biology is the possibility to image biological samples in vitro and in vivo. A special construction of AFM liquid scanner enables to work directly in liquid environments [2]. Artefacts caused by dehydration of samples are eliminated this way. Imaging using AFM is non-destructive and samples can be visualized several times. Physical or chemical fixations as well as coating of surfaces by sputtering for having a better contrast and conductivity are not necessary.
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Corresponding author: email: roman.kubinek@upol.cz, Phone: +420585634285

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Dentin is the basic matter of a human tooth and is formed from an inorganic hydroxyapatite Ca10(PO4)6(OH)2 from approximately 70 %; 20 % of its volume consists of organic material (mostly collagen fibres) and the remaining 10 % is water. Inside the dentin there are dentinal tubules with aperture width ranging from 10 nm to 3 m and being several mm long. Openings of dentinal tubules on the surface are normally covered by enamel or cementum. However, these protective layers can be lost under certain conditions and then fluid flow along these open tubules caused by mechanical, chemical or thermal stimuli can result in uncomfortable pain. Using the standard procedure for preparation of dentin samples in Scanning Electron Microscopy (SEM) a sample is dehydrated in graded acetone series, dried and coated in sputtering device after its fixation. The occurrence of artefacts caused by shrinking cannot be eliminated in practice because dentin is strongly sensitive to dehydration [3]. We have investigated the possibility to examine dentin surface of human teeth under moist conditions using AFM. This is a method that is not based on aggressive chemicals can help in excluding artefacts.

2. Material and methods

Dentin samples have been prepared in the form of discs. Human extracted third molars without decay stored in solution of 0.5 % chloramines for the period of less than one month after their extraction at the temperature of 4 C (according to the rules ISO TR 11 405) have been used. Anatomical crown and apical part of a root have been separated using a diamond disc after removing soft tissue and debris. A 3mm high dentin disc remains after this procedure. Upper surfaces of these discs have been brushed and subsequently polished by paper discs Sof-Lex. However, in many cases a smear layer composed of small particles of dentine and dendritus covers the dentin surface thus preventing it from diagnostics. The smear layer has to be removed using either etching by solutions of acids (e.g., phosphoric acid, polyacrylic acid) or leaving the samples in an ultrasonic cleaning device for several minutes. Every dentin slice is divided into two halves and placed into distilled water in an ultrasonic purified apparatus for 30 minutes. The last method has been found to be more suitable in our research. The samples have then been examined by AFM and SEM. Surface of dentin samples have been observed using AFM and SEM. AFM Explorer manufactured by Veeco-ThermoMicroscopes (USA) has been applied using contact mode with tips from silicon nitride (type 1520-00, Veeco). The imaged surface area has gone from 5 to 100 m and resolution of 300 points per row has been used. Maximum measurable changes of the surface profile have been 10 m. Dentin samples have been stored in distilled water and processed under moist conditions. SEM BS 340 (TESLA, Czech Republic) with magnification going from 10 to 20000 was used. Before observation the samples were dehydrated in acetone, then dried and finally coated by a conductive aurum layer in sputtering unit Polaron E5100.

3. Results
We have firstly paid attention to observation of the clean dentin surface. Figures 1-4 show dentin surfaces and they also demonstrate problems that have been faced using AFM as a method for imaging. The dentin surface with well-shown dentinal tubules is depicted in Fig. 1. Dentinal tubules as well as obvious drafts caused when preparing a sample are shown in Fig. 2. Dentinal tubules surrounded by a wall (caused probably by mechanical preparation of the sample) can be seen in Fig. 3a (AFM image) and Fig. 3b (SEM image). Morphology of dentinal tubules is visualized in 3D image, see Fig. 4.

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Fig. 1 Picture of a dentin surface with opened dentinal tubules without smear layer obtained by AFM (scan range 80x80 m).

Fig. 2 Visible drafts on a surface of a dentin sample caused by mechanical preparation and depicted by AFM (scan range 100x100 m).

Dentinal tubules are surrounded by a wall in this image taken by AFM (scan range 25x25 m)- Fig. 3a and by SEM (bar represents 2 m) Fig. 3b.

Natural curvature of a tooth root in two planes, a small roughness occurring on dentin surface after removing cementum complicated imaging and also could lead to damage of the used tip. This problem has been removed by polishing the surface with fine paper discs of Sof-Lex under water cooling. We had to solve the problem of contamination of sample surfaces. If a surface is not clean enough a lot of strips occur in the image; they are caused by small particles that are moved on a surface by the tip, see Fig. 5a and Fig. 5b. Removal of the smear layer that arises on the surface after its instrumentation and prevents dentinal tubules from imaging is necessary.

Fig. 4 3D-image of dentin surface taken by AFM (scan range 5x5 m).

Fig. 6 Dentin surface with open dentinal tubules as seen by SEM; tubules are mostly covered by smear layer (bar represents 5 m).

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The surface of a dentin sample with tubules is shown in Fig. 6 (SEM image). However apertures of tubules are mostly covered by the smear layer. Removal of the smear layer as well as plugs in dentinal tubules can be safely reached by polishing the samples and subsequently putting them into an ultrasonic bath for 30 minutes. Removal of the smear layer using a 37 % solution of phosphoric acid or combining phosphoric acid with natrium hypochloride leads to a demineralization event. Deproteination of dentin; also the evidence of mechanical instrumentation is present.

Fig. 5a Smear layer on the dentin surface (scan range 50x50 m).

Fig. 5b A lot of strips occur in the image caused by smear layer (scan range 100x100 m).

Dentin surface with apertures of tubules without smear layer under equivalent resolution is depicted in Fig. 7 (AFM image) and Fig. 8 (SEM image).

Fig. 7 The flat dentin surface with opened dentinal tubules without smear layer obtained by AFM (scan range 100x100 m)

Fig. 8 The flat dentin surface with open dentinal tubules as seen by SEM (bar represents 20 m).

Disadvantage of AFM approach is the fact that taking an image requires approx. five minutes. We obtain information about the sample surface just below the tip that covers a small area. Information

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about the rest of a sample surface is missing owing to a small scanned area and this complicates statistical analysis of the surface. Samples have to be moved mechanically below the tip which also causes trouble in a real experiment. To compare with Scanning Electron Microscopy (SEM), this method enables to visualize a large area of the surface and a subsequent enlargement of resolution is possible. We use pictures of surface-dentin morphology obtained by SEM for comparison. Samples have been prepared using a standard method for Scanning Electron Microscopy (dehydration in a grated acetone series, drying in CPD-030, coating by a 5-nm layer of gold and palladium). Observation has been done using microscope SEM Tesla BS 340.

4. Discussion and Conclusions

It is evident from literature that AFM is a frequently used method for imaging of dentin. It is mainly useful in studies of the collagen network of dentin and its changes caused by different chemical agents to dentin [3-4]. Changes in intertubular and peritubular dentin caused by interaction with phosphoric acid, self-etching primers, conditioners and other agents used in bonding of restorative materials can be investigated[5-9]. Also interaction of dentin adhesives with the tooths hard tissues can be observed [10-12]. AFM has been applied in investigations of ultramorphology of superficial and deep dentin and its mechanical properties [13]. Roughness and elasticity of hydrated peritubular and intertubular dentin has been followed. Atomic Force Microscopy enables to observe micro-morphology noncarious cervical lesions as well as functional width of the dentino-enamel junction [14]. This method also provides information about dentin roughness [15] and enables the register of changes in nanomechanical properties of dentin during its storage.We would like to note at this point that AFM has also some limitations. An inevitable use of a cantilever results in many difficulties and restrictions in measurements and sample preparation. Furthermore, a high cost of AFM systems prevents their widespread industrial and clinical use, and they are difficult to operate in a laboratory that does not specialize in AFM technology [16]. When a tip scans across a sample it induces a dynamic interaction force between the tip and the surface. The dynamic behaviour is complicated and a precise analysis is difficult, but it can influence resolution of the surface image [17]. According to our experience AFM method is not suitable for the estimation of properties of larger areas on a surface. It is time-consuming and requires a flat surface that is accessible to a tip of an AFM microscope. This means a rather strong restriction to flatness of a sample as a whole as well as that concerning flatness of its detailed parts. It is without doubts that AFM brings new possibilities in imaging of dentin surfaces. For this reason, further spreading of this non-destructive method is expected in the near future. We can see its main advantage in the fact that this method enables the study of wet and chemically non-modified surfaces and so it does not face trouble connected with artefacts caused by dehydration as the other methods do.
Acknowledgements: This work is supported by the projects of the Czech Ministry of Educations 1M0512 and MSM6198959216

References
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