Compensating thickness effects in micro X-ray fluorescence spectroscopy using integrated optical microscopy for...

A non-invasive method using the integrated optical microscope of a laboratory-based 2-dimensional micro X-ray fluorescence spectroscopy (2D µXRF) instrument to determine the thickness of soft matter samples has been successfully developed, validated, and applied. This easy-to-use method is applicable to soft matrices in a thickness range from 25 to 1000 µm. The main advantage of this method is that thickness determination is directly related to the physical thickness of the sample, rather than its optical thickness, and it does not affect the sample structure, i.e., it does not require any drying or embedding. Elemental composition and distribution analysis by 2D µXRF can be performed on the same sample specimen, using the same setup directly before or after thickness determination. Knowledge of the thickness of different samples can be used to normalize elemental intensities to compensate for the mass-thickness effect, enabling reliable comparison of samples of different thicknesses. By systematically probing several points on a sample surface, this approach can also be used to correct the elemental intensity of unevenly thick soft matter samples. The aspect of correcting element intensities based on the determined thickness should enable direct quantification of element contents using µXRF and external calibration in future work.