Intestinal stem cells (ISCs) continuously generate new daughter cells to maintain intestinal function; during aging, ISC function declines and causes intestinal dysfunction. Mitochondria are a powerhouse of the cell, and their morphology changes during stem cell differentiation, suggesting that mitochondrial morphology is associated with stem cell behavior. However, if age affects mitochondrial morphology in ISCs remains unclear. In this study, we used the UAS-Gal4 system combined with immunostaining and found that mitochondria were more fragmented in aged ISCs. To improve the resolution of mitochondrial imaging for rigorous analysis, we utilized the expansion microscopy method to expand ISCs about 2.5-fold in combination with mitochondrial immunofluorescence labeling. Our results showed that ISCs could be expanded without disrupting mitochondrial structure. With this simple and inexpensive method for nanoscale optical imaging of mitochondria, in the future we can better understand the effects of age on mitochondrial morphology, and probably mitochondrial cristae structure, in stem cells, and hopefully have in-depth investigation and medical applications.