High-Resolution Imaging Methods for Identification of Calcium Crystal Types in Osteoarthritis

Abstract

The detection and differentiation of BCP and CPP crystals in calcified tissue is an important factor in the context of research and potential future treatment of osteoarthritis and chondrocalcinosis. Current standard methods originate from clinical practice and often lack precision in the correct identification of the calcium crystal type. In this work, a step-by-step guide for the use of the high-resolution imaging methods of tissue sections, Raman spectroscopy and scanning electron microscopy (SEM) in combination with energy-dispersive X-ray spectroscopy (EDS), for calcium crystal identification is presented. Sample preparation including Von Kossa staining, measurement and measurement parameters, data processing and data analysis methods are discussed and described. Furthermore, the different methods are compared to show advantages and disadvantages. Overall, Raman spectroscopy is a reasonable method from an economic point of view and regarding the time/effort required for acquiring highly reliable data in calcium crystal identification. Potentially, semi-quantitative results can be obtained with little effort and without the destruction of the respective test sample. The analysis/penetration depth during the Raman measurements, which is not precisely defined, poses a potential problem for accuracy. SEM can also be used for this task but requires more time, advanced technical knowledge and a pre-treatment of the samples using, e.g., gold sputtering, which may distort further analysis on the specific specimen. Therefore, this technique yields additional value compared to Raman spectroscopy only with additional research questions needed to be answered in the same sample, such as analysis of the sample topography or analysis of other unknown particles/deposits using EDS. The methods described in this manuscript are helpful for retrospective analyses in the context of research, but can also be used for potential future treatment strategies to discriminate between osteoarthritis and chondrocalcinosis patients.