Human dental enamel is organized by prisms that are structured between 3 and 6 µm in diameter. Determining the relationships between different treatments on the surface of enamel using ultrastructural analysis is the purpose of many in vitro experiments. Different sample pretreatments have been reported in the literature. Grinding and polishing are common procedures for enamel preparation. They provide a flat and standardized surface, which is imperative for the use of some techniques such as ATR‐FTIR. However, for morphological analysis, SEM and AFM represent easier methods to measure and reduce the biological sample variation. Therefore, the objective of this study was to establish how different forms of enamel preparation can influence the advent of artifacts during ultrastructural observation, especially by AFM analysis. Four groups (n = 10) were tested: (a) without preparation; (b) polishing with a diamond paste; (c) grinding with decreasing granulations of silicon carbide papers; (d) grinding with polishing. Images were obtained using the Peak‐Force Tapping mode. After the first images were obtained, all fragments were acid etched with 37% phosphoric acid for 30 seconds, rinsed for 60 seconds, and dried intensively. Upon grinding and polishing, the exposure of the inner enamel surfaces provided a less mineralized layer that was marked by scratches and a higher susceptibility to treatments. Moreover, using native enamel provided more valuable information on the surface and the roughness changes for clinical applications. In addition, phosphoric acid is an option for observing the prismatic arrangement after grinding and/or polishing changes the morphology. Research Highlights The use of native enamel samples to investigate the effects of different treatments on surface should be preferred in research, when the technique allows it.

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