Super‐Resolution Axial Imaging for Quantifying Piconewton Traction Forces in Live Cells

Abstract Cell mechanics play a pivotal role in regulating numerous biological processes. Although super‐resolution microscopy enables the imaging of cellular forces in the lateral dimension with sub‐10‐nm resolution, achieving comparable resolution along the axial dimension remains a significant challenge. Here, we introduce metal‐induced energy transfer (MIET)‐based tension probe microscopy (MIET‐TPM), a technique for mapping cellular mechanical forces with nanometer precision in the axial direction. This approach combines the nanometer spatial resolution of MIET imaging with the piconewton sensitivity of DNA‐hairpin‐based molecular tension probes (MTPs), enabling the simultaneous observation of both the plasma membrane and force‐exerting molecules in the axial dimension. Using MIET‐TPM, we mapped axial integrin tension within focal adhesions and podosomes, alongside their corresponding plasma membrane height profiles, offering detailed insights into the nanoscale structures and mechanisms involved in force transmission. Notably, MIET‐TPM can be implemented on any fluorescence microscopy setup without hardware modifications, making it a versatile and accessible tool that promises to become an integral part of future cellular mechanobiology analysis.