## 4 Discussion  
Micro-CT has become an important tool for rapid and nondestructive high-resolution 3D imaging in biology. Relying on modern X-ray source technology and precision machinery and detection systems, people have constantly pursued higher pixel numbers and ultrahigh spatial resolutions. At present, the resolution of X-ray CT technology has reached 30-50 nm [25; 26; 27]. Compared with the improvement of the pixels and the resolution, X-ray CT technology has not made much progress in terms of improving the imaging contrast [28]. For weakly absorbing substances such as fat and muscle in biological tissues, the ideal absorbing contrast is often not achieved in high-pixel and high-resolution imaging. The purpose of this experiment was to establish the optimal micro-CT-scanning method of zebrafish adipose tissue. We first compared the scanning effects of zebrafish adipose tissue under 4000 and 2000 pixels. In the experiment, the contrast between zebrafish adipose tissue and non-adipose tissue was the best under 2000 pixels, which was convenient for later image processing and quantitative calculation of adipose tissue. In addition, to improve the absorbing contrast of adipose tissue, low-energy X-rays were used in the scan. Zebrafish bone tissue is much denser than adipose tissue. When using low-energy X-rays for whole-fish scanning, the bone tissue is extremely beam-hardened due to high X-ray absorption, and black artifacts arise around the bone tissue, which seriously affect the subsequent image processing and quantitative calculation of related parameters. This situation is commonly seen in CT scans of metal implants in orthopedics [29]. Scanning the sample at 360\({}^{\circ}\) is one effective method to remove scanning artifacts. In this experiment, when scanning at 180\({}^{\circ}\) at 2000 pixels, the X-ray was blocked by the bone with high-density, as it could not penetrate the bone, which affected the X-ray imaging and led to the formation of black artifacts. However, in the reconstructed image scanned at 360\({}^{\circ}\), although the X-ray was also blocked by the bone, the missing data from 0 to 180\({}^{\circ}\) were supplemented in the scanning from 180 to 360\({}^{\circ}\), which generated more complete data and reduced black artifacts. The boundary between bone tissue and other tissue was clear, which was conducive to image processing and the quantitative calculation of adipose tissue. Figure 13: Distribution of abdominal adipose tissue thickness (**A**) and 3D surface rendering (**B**). From the CT scan of the zebrafish adipose tissue, it was found that there were adipose tissues in the head, abdominal cavity, subcutaneous area, and back of the zebrafish. It is generally believed that the main accumulation sites of fish adipose tissue are the abdominal cavity, liver, and muscle. It is common in fish nutrition research to assess the fat content of fish by detecting the fat content of its abdominal cavity, liver, and muscle [30; 31]. We also found fat under the skin of the back and both sides of the body of the zebrafish, indicating that the subcutaneous area is the main accumulation site of zebrafish adipose tissue, and thus, that the accumulation of subcutaneous adipose tissue in fish cannot be ignored. The accumulation site and content of adipose tissue are closely related to the physiology, biochemistry, and energy metabolism of the animal. Adipose tissue in different sites has different physiological functions in humans and mammals [32; 33; 34], but the distribution of fish adipose tissue has not yet been clarified. The CT imaging method in this experiment can intuitively reflect the distribution of adipose tissue in the fish, which will be helpful in research on the physiology, biochemistry, and energy metabolism of various fish species. The zebrafish head also has notable adipose tissue, showing that fat is abundant in the brain of the zebrafish, which is not surprising. Animals, including fish, have many phospholipids in the brain.