Organoid models are becoming increasingly popular in biological research and screening due to their ability to reproduce the complexity of real tissues. In order to simulate the human lung organs in vivo, we cultured primary human lung epithelial cells under conditions conducive to 3D structure formation, reproducing the morphological and functional characteristics of the lung airway. In lung organoid culture, epithelial stem cells and progenitor cells are cultured in ECM supplemented with a series of growth factors. The organoids then grow into complex structures, preserving clusters of multilineage epithelial cells. These characteristics make organoid culture a promising approach that can be widely applied in basic and translational methods, such as drug screening and disease modeling. Here we describe a high connotation imaging method that can monitor and visualize the growth and differentiation of lung organoids, reconstruct 3D structures, analyze complex organoid structures, cell morphology and vitality, as well as the expression of different cell markers. 3D lung organoids implanted in Matrigel domes. Developmental organoids include spherical objects with complex shapes, including cavities and bladder structures. During the 8-week growth process, we observed an increase in size and complexity. Use transmission light to monitor the organs, and then use 20-30 layers of Z-axis scanning to image the stained organs through the matrix gel. The objective lens is 10 – 40x. Use ImageXpress ® The newly configured laser light source of Confocal HT.ai intelligent confocal high content imaging analysis system significantly improves the illumination intensity, thereby significantly enhancing brightness, detection sensitivity, and imaging speed.