Microsc. Microanal. 20, 937–945, 2014 doi:10.1017/S1431927614000622 © MICROSCOPY SOCIETY OF AMERICA 2014 Comparative Study of the Measurement of Enamel Demineralization and Remineralization Using Transverse Microradiography and Electron Probe Microanalysis Nathan J. Cochrane, 1,✠ Youichi Iijima, 2 Peiyan Shen, 1 Yi Yuan, 1 Glenn D. Walker, 1 Coralie Reynolds, 1 Colin M. MacRae, 3 Nicholas C. Wilson, 3 Geoffrey G. Adams, 1 and Eric C. Reynolds 1, * 1 Oral Health CRC, Melbourne Dental School, Bio21 Institute, The University of Melbourne, 720 Swanston Street, VIC 3010, Australia 2 Department of Oral Health, Nagasaki University Graduate School of Biomedical Sciences, Unit of Social Medicine, 1-7-1 sakamoto, Nagasaki 852-8588, Japan 3 Microbeam Laboratory, CSIRO Process Science and Engineering, Bayview Avenue, Clayton, VIC 3168, Australia Abstract: Transverse microradiography (TMR) and electron probe microanalysis (EPMA) are commonly used for characterizing dental tissues. TMR utilizes an approximately monochromatic X-ray beam to determine the mass attenuation of the sample, which is converted to volume percent mineral (vol%min). An EPMA stimulates the emission of characteristic X-rays from a variable volume of sample (dependent on density) to provide compositional information. The aim of this study was to compare the assessment of sound, demineralized, and remineralized enamel using both techniques. Human enamel samples were demineralized and a part of each was subsequently remineralized. The same line profile through each demineralized lesion was analyzed using TMR and EPMA to determine vol%min and wt% elemental composition and atomic concentration ratio information, respectively. The vol%min and wt% values determined by each technique were significantly correlated but the absolute values were not similar. This was attributable to the complex ultrastructural composition, the variable density of the samples analyzed, and the nonlinear interaction of the EPMA-generated X-rays. EPMA remains an important technique for obtaining atomic ratio information, but its limitations in determining absolute mineral content indicate that it should not be used in place of TMR for determining the mineral density of dental hard tissues. Key words: transverse microradiography (TMR), electron probe microanalyzer analysis (EPMA), remineralization, demineralization I NTRODUCTION Dental caries is the prevalent disease process whereby net mineral is lost from the hard tissues of the teeth (Selwitz et al., 2007). New approaches to promote the net uptake of mineral back into these damaged areas are currently receiving much research interest (Cochrane et al., 2010). A variety of different methods can be used to measure this loss or gain of dental mineral including transverse microradiography (TMR), polarized light microscopy, microhardness determination, and electron probe microanalysis (EPMA) with energy- dispersive or wavelength-dispersive spectrometry (ten Bosch & Angmar-Mansson, 1991). TMR is the standard method for the measurement of changes in mineral content of the dental hard tissues (ten Bosch & Angmar-Mansson, 1991). It utilizes a monochromatic X-ray beam to determine mass attenuation which is then converted to volume percent mineral (vol%min). EPMA can be used for the chemical analysis of samples with a high spatial resolution yielding both qualitative identification of elements and quantitative compositional information of microvolumes (Landis, 1979). EPMA analysis is based on the emission of characteristic X-ray radiation of elements within a small volume of sample excited by the impact of a focused beam of electrons (Chu et al., 1989; Heinrich & Newbury, 1991) whose energies and relative abundance depend upon the composition of the sample. EPMA has been used for the analysis of dental tissues since the technique was first introduced (Boyde et al., 1961). At this time it was appreciated that biological samples of variable density were difficult to analyze quantitatively (i.e., determine their elemental composition) such that it was described as a semi-quantitative technique (Boyde et al., 1961). However, more recently, investigators have been using EPMA for quantifying the mineral content of dental hard tissues of variable density by measuring the wt% composition of the major elements in the tissue and imply- ing that their change indicates that demineralization or remineralization has occurred (Yip et al., 1995; Ngo et al., 2006; Ab-Ghani et al., 2007; Baroni & Marchionni, 2011). This approach has been studied by Ngo et al. (1997) who found that there was a strong correlation in the mineral *Corresponding author. e.reynolds@unimelb.edu.au ✠ We dedicate this work to the memory of Nathan J. Cochrane our friend and colleague. His dedication and passion for cariology was unmatched; his energy, enthusiasm and friendship will be sorely missed. Revised June 28, 2013; accepted February 26, 2014