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Abstract Recent developments such as multi-harmonic Atomic Force Microscopy (AFM) techniques have enabled fast, quantitative mapping of nanomechanical properties of living cells. Due to their high spatiotemporal resolution, these methods provide new insights into changes of mechanical properties of subcellular structures due to disease or drug response. Here, we propose three new improvements to significantly improve the resolution, identification, and mechanical property quantification of sub-cellular and sub-nuclear structures using multi-harmonic AFM on living cells. First, microcantilever tips are streamlined using long-carbon tips to minimize long-range hydrodynamic interactions with the cell surface, to enhance the spatial resolution of nanomechanical maps and minimize hydrodynamic artifacts. Second, simultaneous Spinning Disk Confocal Microscopy (SDC) with live-cell fluorescent markers enables the unambiguous correlation between observed heterogeneities in nanomechanical maps with subcellular structures. Third, computational approaches are then used to estimate the mechanical properties of sub-nuclear structures. Results are demonstrated on living NIH 3T3 fibroblasts and breast cancer MDA-MB-231 cells, where properties of nucleoli, a deep intracellular structure, were assessed. The integrated approach opens the door to study the mechanobiology of sub-cellular structures during disease or drug response. 

Mechanical properties play important roles at different scales in biology. At the level of a single cell, the mechanical properties mediate mechanosensing and mechanotransduction, while at the tissue and organ levels, changes in mechanical properties are closely connected to disease and physiological processes. Over the past three decades, atomic force microscopy (AFM) has become one of the most widely used tools in the mechanical characterization of soft samples, ranging from molecules, cell organoids and cells to whole tissue. AFM methods can be used to quantify both elastic and viscoelastic properties, and significant recent developments in the latter have been enabled by the introduction of new techniques and models for data analysis. Here, we review AFM techniques developed in recent years for examining the viscoelastic properties of cells and soft gels, describe the main steps in typical data acquisition and analysis protocols, and discuss relevant viscoelastic models and how these have been used to characterize the specific features of cellular and other biological samples. We also discuss recent trends and potential directions for this field.