Studying mechanobiology is increasing of scientific interests in life science and nanotechnology since its impact on cell activities (e.g., adhesion, migration), physiology, and pathology. The role of apical surface (AS) and basal surface (BS) of cells played in mechanobiology is significant. The mechanical mapping and analysis of cells mainly focus on AS while little is known about BS. Here, high‐speed scanning ion conductance microscope as a powerful tool is utilized to simultaneously reveal morphologies and local elastic modulus (E) of BS of genotype‐defined metastatic intestinal organoids. A simple method is developed to prepare organoid samples allowing for long‐term BS imaging. The multiple nano/microstructures, i.e., ridge‐like, stress‐fiber, and E distributions on BS are dynamically revealed. The statistic E analysis shows softness of BS derived from eight types of organoids following a ranking: malignant tumor cells > benign tumor cells > normal cells. Moreover, the correlation factor between morphology and E is demonstrated depending on cell types. This work as first example reveals the subcellular morphologies and E distributions of BS of cells. The results would provide a clue for correlating genotype of 3D cells to malignant phenotype reflected by E and offering a promising strategy for early‐stage diagnosis of cancer.
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