The distinction between biological processes of adipose tissue expansion is crucial to understanding metabolic derangements, but a robust method for quantifying adipocyte size has yet to be standardized. Here, we compared three methods for histological analysis in situ: one conventional approach using individual micrographs acquired by digital camera, and two with whole-slide image analysis pipelines involving proprietary (Visiopharm) and open-source software (QuPath with a novel ImageJ plugin). We found that micrograph analysis identified 10–40 times fewer adipocytes than whole-slide methods, and this small sample size resulted in high variances that could lead to statistical errors. The agreement of the micrograph method to measure adipocyte area with each of the two whole-slide methods was substantially less (R² of 0.6644 and 0.7125) than between the two whole-slide methods (R² of 0.9402). These inconsistencies were more pronounced in samples from high-fat diet fed mice. While the use of proprietary software resulted in the highest adipocyte count, the lower cost, ease of use, and minimal variances of the open-source software provided a distinct advantage for measuring the number and size of adipocytes. In conclusion, we recommend whole-slide image analysis methods to consistently measure adipocyte area and avoid unintentional errors due to small sample sizes.

脂肪组织扩张的生物学过程之间的区别对于理解代谢紊乱至关重要，但定量脂肪细胞大小的有效方法尚未标准化。在这里，我们比较了三种原位组织学分析方法：一种使用数码相机获取的单个显微照片的常规方法，另外两种使用涉及专有（Visiopharm）和开源软件（带有新颖ImageJ插件的QuPath）的全幻灯片图像分析管道。我们发现，显微照片分析确定的脂肪细胞比全玻片方法少10–40倍，而这种小样本量导致高方差，这可能导致统计误差。显微方法来测量与每个所述两个全滑动方法脂肪细胞区域中的协议是显着更少（R ²的0.6644和0.7125）比两个全滑动方法之间（R ²0.9402）。这些不一致在高脂饮食喂养小鼠的样品中更为明显。尽管使用专有软件导致了最多的脂肪细胞计数，但是开源软件的较低成本，易用性和最小差异为测量脂肪细胞的数量和大小提供了明显的优势。总之，我们建议使用全幻灯片图像分析方法来一致地测量脂肪细胞面积，并避免由于小样本量造成的意外错误。