Background

The role of small animal models in basic science research has rapidly expanded amidst the emergence of novel quantification techniques [1]. Precision measurement in such models has endeavored to unearth underlying mechanisms of action in estrogen-receptor signaling and innovative modes of drug delivery for quality improvement [2,3]. Genetically modified mice have an indispensable purpose in assessing the role of specific genes in key physiological functions and phenotypic changes that are often measured through histological analysis [4,5]. Nonetheless, such models may fail to provide either pertinent longitudinal data, an important consideration for examining structural development (fat, muscle, bone), or pathological changes in tissues such as lung fibrosis.

As an alternative approach to examining in vivo features at a fixed point in time, non-invasive techniques that allow for periodic imaging over time can quantitatively measure varied physiologic and pathologic phenotypes [5]. The benefits of live imaging of murine structures include quantifiable measurements for organic tissue-dependent changes at specific growth stages without inflicting harm or interruption to the life cycle. Non-invasive tools also enhance experimental designs by yielding a series of data points over the entire study duration, limiting the overall cohort size needed, and allowing more precise measures of inter-animal variability [6].

One of the most frequently utilized tools for diagnostic imaging is X-ray computed tomography (CT), which relies on employing X-ray beams in direct contact with the organism to create a cross-sectional tomographic representation. Hounsfield units (HU) is a relative quantitative measurement of radio density used in the interpretation of computer tomography images in the linear scale; the radio density of distilled water at a standard temperature and pressure is defined as 0 HU, whereas the radio density of air is -1,000 HU. A phantom with a known radio density calibrates machines, ensuring they provide the correct HU. Most animal tissues do not exceed +2,000 HU and standard clinical limits range from -1,000 to +3,071 [7].

In pre-clinical research, the ability of the micro-CT scanner to elicit information on body composition comes highly appreciated, enhancing surveillance of disease development, classification, and ramifications [8]. Whether analyzing bone mineral density for studying osteoporosis, distinguishing airspace changes for characterizing lung fibrosis, or comparing adipose volume change, non-invasive in vivo imaging using micro-CT scanners enables high spatial resolution with longitudinal monitoring [9].

Without a standardized protocol for utilization, not only does the reproducibility of research suffer, but it also hinders the ability to build upon the ever-evolving field of structural analysis and to develop an eventual gold standard for body composition characterization. Simple method articles serve as valuable resources for researchers who wish to incorporate micro-CT into their studies.

In this article, we propose an easy-to-follow guide for micro-CT users to reconstruct the physical structure of mice to obtain standard non-invasive imaging in mice-based long-term experiments. Our protocol improves upon existing methods by providing accessible, step-by-step instructions that minimize the need for specialized training. It simplifies workflows into manageable tasks, promotes standardization with many visual aids and troubleshooting guidance, and improves reliability. This protocol details a reproducible manner by which researchers can precisely attain data on lean tissue, adipose, bone structure, bone mineral density, and airspace quantification in the lung, enabling the investigation of gene-based translational applications.