Influence of nanoendoscopy AFM imaging of intracellular structures on cell proliferation and stress response

The recent development of nanoendoscopy atomic force microscopy (NE-AFM) has enabled direct imaging of nanodynamics within living cells. However, this technique involves repeated nanoneedle tip insertions or 2D scans of the inserted tip, raising concerns about its impact on cellular viability and function. Although previous fluorometric assays indicated no lethal damage to cells, the potential effects on cellular functions remain unclear. To address this issue, we have investigated the influence of 2D/3D NE-AFM imaging on cell proliferation and calcium stress responses. Our findings reveal that typical 2D/3D NE-AFM imaging conditions do not significantly affect cell division intervals. For calcium stress responses, 2D imaging with a scan size smaller than 1 μm minimally induces calcium responses, whereas 3D imaging triggers transient calcium responses at the beginning of the scan. These responses stabilize within ∼15 minutes, allowing intracellular calcium levels to return to baseline for the remaining imaging period. This study contributes to establishing conditions for NE-AFM imaging with a minimal impact on the cell functions, facilitating accurate interpretation of the obtained results, and advancing our understanding of various intracellular nanodynamics.