Inorganic elements have a variety of functions in living organisms. Essential elements such as zinc (Zn) and iron (Fe) play critical roles in biological processes and an optimal level is needed for proper function. Non-essential, potentially toxic elements, such as lead (Pb) and arsenic (As), have no biological function, and exposures to these cause adverse health effects.(1,2) The acute and chronic health implications of potentially toxic elemental exposures underlie the need to collect biomarkers of exposure.(1) Typically, biomarkers for exposure to inorganic elements reflect only recent exposures, such as blood (hours-days), urine (several days), hair (months), or toenails (months), with blood being the most common biomarker used in exposure studies (3–5). The half-life of many elements in these tissues is relatively short, on the order of days to months (6–9). There are fewer biomarkers of cumulative exposure to inorganic elements over much longer periods which would be better biomarkers in epidemiological studies of chronic health outcomes. Pb in bone and cadmium (Cd) in urine are two such examples. Biomarkers of early life exposures not influenced by recent exposures are more challenging to collect. These types of biomarkers are particularly valuable for studies of the developmental origins of health and disease in adults, an area of growing interest (3,10,11).

Teeth, which accumulate inorganic elements during active growth and development, have been used in health studies as a means to capture exposures ranging from fetal to adolescent life stages (10,12). Early studies of teeth used ground, whole teeth to measure elemental or radioisotope accumulation (11,13,14). For this study, we are particularly interested in tooth enamel which is primarily composed of hydroxyapatite crystal mineral.(15) The mineralization process in tooth enamel occurs between 3 months to 16 years of age depending on the individual tooth development.(16,17) Upon completion of enamel, the elements and isotope ratio inside the enamel matrix remain unchanged throughout the lifetime.(10) It makes tooth enamel an appropriate biomarker to assess the diet, elemental exposure, and even geographical origin of the subject during the developmental window unlike other biomarker like bone which typically undergoes several reformations during adulthood.(18) (3,10,12). Thus, information on the timing of exposure could potentially reveal how exposures to inorganic elements during critical developmental windows affect later life health outcomes (3,10,12).

Recent studies have used laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) of deciduous teeth (3,10,12,19,20). LA-ICP-MS has been used on teeth sections to obtain a record of exposure relative to the day of birth (21,22). However, the spatial resolution of this technique is currently limited by the laser to 4 μm resolution at the lowest, with most operating at 10–20 μm (21). Fine-resolution assessment using synchrotron x-ray fluorescence (SXRF) could be used to assess exposure levels down to a daily basis or below. The resolution of SXRF, although dependent on the beamline, can be on the order of 0.3 μm (23). SXRF may provide the opportunity to disentangle complex exposures with higher resolution, which will aid studies of cyclic metabolic processes, saving time by mapping faster than typical LA-ICP-MS (19,20). SXRF methods have been used effectively for elemental mapping of tissue and teeth in other studies (24–26).

In addition, SXRF has the advantage of using a calibration that does not require matrix-matched composition for the quantification of elements, whereas LA-ICP-MS typically requires matrix-matched controls to verify quantitative units. Around the world, SXRF systems vary considerably in terms of spatial resolution and limit of detection capabilities. In this study, we used the 2-ID-E Microprobe at Advanced Photon Source at Argonne National Lab, which has a sub-micron spatial resolution. High-resolution SXRF is done at large facilities, typically at national labs in the US, that now use micrometer spatial resolution with high enough intensity to measure elemental exposures in teeth (24–26). Importantly, LA-ICP-MS has the advantage of accessibility through in-lab measurements, whereas SXRF typically requires beamline access through a typically long application and review process which can hinder accessibility greatly. LA-ICP-MS, as a lab-based method, is much easier for novel sample measurements with an ever-increasing number of labs acquiring the technology. Finally, detection limits for each methodology can vary. LA-ICP-MS for a single pixel of data will be able to detect ppm concentrations. SXRF will vary based on the synchrotron used to collect data, the detectors used, and the spatial resolution, but the samples measured here would have 0.1 ppm level detection limits.

In this study, we compared LA-ICP-MS and SXRF for the measurement of inorganic elements in teeth, which were imaged to identify pre- and post-natal exposure periods in the enamel. We demonstrate the similarities of overall elemental quantification within the measured areas of the tooth and discuss the advantages and disadvantages of each technique.

This study sought to compare two elemental imaging methods for the measurement of teeth as retrospective biomarkers of inorganic element exposure and demonstrated the feasibility of using SXRF to evaluate elemental distributions in teeth. SXRF was compared to standard LA-ICP-MS. We found the SXRF is comparable to LA-ICP-MS for quantification of elements and demonstrated agreement between SXRF in μg/cm² and calcium-normalized LA-ICP-MS data over pre- and post-natal areas. Quantification between LA-ICP-MS and SXRF was comparable, with both methods displaying the ability to measure the concentrations of a suite of elements in the tooth.

SXRF data over the enamel areas in the teeth had close relationships with the quantification from LA-ICP-MS data. This was despite the key differences in the sampling methodology. SXRF should measure slightly deeper into the tooth, whereas LA-ICP-MS will take only a small amount of material from the surface for measurement –estimated as 1 μm depth per second at 75% power. The escape depth for even the highest energy characteristic x-rays (Pb) of our elements of interest would represent an average depth of 0.2 mm (200 μm), as taken from NIST attenuation coefficients (27). For other elements in the XRF spectra with characteristic energies of 5 keV or less, the average penetration depth would be 20 μm according to NIST attenuation coefficients (27). This would produce a bias error in comparison to the LA-ICP-MS data, which is all measured at the same depth. Ideally, this difference in depth for SXRF should not cause issues in quantification since calibration accounts for these changes based on energy and penetration depth. Nonetheless, we found no differences in our quantification based on sample thickness.

The elements we chose for comparison represent those that were easily measured by both methods. Certain elements of interest, like arsenic or mercury for example, were not accurately quantified on LA-ICPMS, whereas an element like strontium was not included in our SXRF analysis because the beamline energy was set at 15.8 keV which would not have been enough energy to excite the strontium K-line with an initial binding energy of 16.1 keV. Future work should focus on more samples with direct comparisons of more variability of elements that were unable to be detected in this work, and, thus, not compared between the methods.

This study did have some limitations, which should be addressed in future work. First, the sample size was very small with only three total sample measures. Despite this, measurements on a micrometer scale provided plenty of data points for comparisons and validation of methodology, and this is important for the use of both techniques in future studies. The findings shown here support the use of both SXRF and LA-ICP-MS in future exposure assessment studies of teeth and health outcomes. SXRF and LA-ICPMS both have different advantages. Specifically, SXRF teeth exposure assessment has the advantages of being non-destructive and having finer spatial resolution, but the disadvantage of being more challenging to access, and elemental accuracy dependent on beam properties. LA-ICP-MS has the major advantage of being a benchtop instrument but can have less sensitivity for certain elements (anything lighter than Be).

Overall, SXRF and LA-ICP-MS are both suitable for 2D spatial exposure assessment in teeth. SXRF offers some significant advantages over LA-ICP-MS in non-destructive measures, and lower spatial resolutions, but has the significant disadvantage of needing to be at a synchrotron source for measurement. Although the lab-based 2D x-ray fluorescence approach is on the rise, it may open up the technology to become more widely available in lab settings. In the future, more studies utilizing SXRF would benefit the epidemiological evidence of finer temporal exposures and their impacts on health or metabolic cycles.