Protocols for preparation of biomineralized samples for computed tomography

Abstract

Computed tomography spans a versatile set of techniques that range several length scales and modalities. It is common to them all that the sample must be prepared in a way which allows for addressing the scientific question(s) posed as well as being suitable for the specific setup of the experiment. We present two lathe-based sample preparation workflows developed to prepare biomineralized samples (here bone) for two very different experiments in terms of setup, types of questions asked, and sample requirements. The first experiment, involving the measurement of high throughput (synchrotron) micro-computed tomography, required the preparation of many samples with homogeneity in size, shape, and bone site. This was achieved through a particular sequence of cutting and embedding steps finalized by lathe milling. The resulting samples were cylindrical in shape with diameters close to the field of view of the ensuing tomography experiment, which allowed maximizing the investigated sample volumes. The second experiment was a combined ptychography and X-ray fluorescence nano-computed tomography experiment, which required preparation of a few-micrometer-sized sample. Moreover, the scientific interest was in a specific, localized feature in bone. Thus, the sample had to be extracted from a precise location from within the whole bone. Again, the developed workflow comprised many steps, including both lathe milling and focused ion beam milling. Importantly, localized preparation was enabled by measuring in-house X-ray micro-computed tomography at crucial points in the workflow. The presented workflows provide examples of preparation pathways that can be standardized and strongly increase the throughput, quality, and success rate of tomography experiments.