3D nanomechanical mapping of subcellular and sub-nuclear structures of living cells by multi-harmonic AFM with long-tip microcantilevers

Yuri M Efremov,Arvind Raman,Daniel M Suter,Peter S Timashev

doi:10.1038/s41598-021-04443-w

Yuri M Efremov, Arvind Raman + Show 2 more

Open Access

https://doi.org/10.1038/s41598-021-04443-w

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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.

Highlights

Changes in the mechanical properties of whole living cells are known to be closely related to a large variety of physiological and pathological p rocesses[1,2,3,4,5,6]
We have recently developed a multi-harmonic dynamic Atomic Force Microscopy (AFM) method to map quantitatively the nanomechanical properties of soft biological samples in a liquid environment at kHz frequencies[29,30,31,32], which is compatible with commercial AFM systems equipped with a direct-excitation setup
We will focus on the implementation that uses deflection feedback (0th harmonic feedback) on living cells

Summary

Introduction

Changes in the mechanical properties of whole living cells are known to be closely related to a large variety of physiological and pathological p rocesses[1,2,3,4,5,6]. Several new techniques have recently emerged to characterize the mechanical properties of the subcellular components directly inside living cells. Deeper below the cell surface including cytoskeletal elements, nucleus, mitochondria, lysosomes, and Golgi apparatus generally remains unavailable To address this challenge, many efforts have been made recently to image sub-surface features through tipgenerated stress or electric fields in AFM19–21, some of which might be applied to the living cells[22,23,24,25]. Subcellular features have often been observed with Peak Force T apping[26,27] or d AFM28,29 methods These prior techniques are generally limited either in spatial, or temporal resolution while imaging live cells or are unable to quantify the properties of the sub-cellular object being imaged

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