Atomic force microscopy (AFM) is a powerful, multifunctional imaging platform that allows biological samples, from single molecules to living cells, to be visualized and manipulated. Soon after the instrument was invented, it was recognized that in order to maximize the opportunities of AFM imaging in biology, various technological developments would be required to address certain limitations of the method. This has led to the creation of a range of new imaging modes, which continue to push the capabilities of the technique today. Here, we review the basic principles, advantages and limitations of the most common AFM bioimaging modes, including the popular contact and dynamic modes, as well as recently developed modes such as multiparametric, molecular recognition, multifrequency and high-speed imaging. For each of these modes, we discuss recent experiments that highlight their unique capabilities.

Mechanobiology focuses on how physical forces and the mechanical properties of proteins, protein assemblies, cells and tissues contribute to signalling, development, cell division, differentiation and sorting, physiology and disease 1-4. On virtually any scale, ranging from organisms 2,4 to components such as organs 5,6 , tissues 3,7 , cells 8-10 , viruses 11,12 , complex extracellular or intracellular architecture (including vesicles, the extracellular matrix or actin network 13,14) or single proteins 15-17 , biological systems respond to mechanical forces and generate mechanical cues. In mechanobiology, living systems are described by cycles of mechanosensation, mechanotransduction and mechanoresponse 2,18. In addition to its state, the functional response of a living system depends on the nature of the mechanical signal, whether it is applied at the nanometre or micrometre scale, for a short or long time, with low or high magnitude, and on whether it is scalar or vectorial. Nanotechnological and microtechnological approaches have enabled tremendous progress in quantifying the mechanical properties of biological systems. The links between mechanical response, morphology and function, however, are conspicuously ill understood. The most widely used approaches to structurally map the mechanical properties and responses of biological systems, ranging from millimetre to sub-nanometre resolution and from micronewton to piconewton sensitivity, are based on atomic force microscopy (AFM) 19,20. In this Review, we survey the exciting developments in AFM-based approaches towards the morphological mapping of a wide variety of mechanical properties and the characterization of the functional response of biological systems under physiologically relevant conditions. We further discuss key challenges and caveats that have to be taken into account to overcome the limitations of AFM-based approaches to more fully describe the mechanical properties of living systems and highlight how complementary techniques can contribute to directly linking the functional responses of complex biological systems to mechanical cues. Characterizing biosystems by AFM The introduction of AFM in 1986 opened the door to imaging and manipulating matter at the atomic, molecular and cellular scales and was central to the nascent nanotechnological revolution 21,22. Of particular importance for the characterization of biological systems, atomic force microscopes can operate in aqueous environments and at physiological temperatures. In an atomic force microscope, a cantilever that is several micrometres long and has a molecularly sharp probe at the end is used to trace the sample topography, detecting

We report a microarray of cantilevers to detect multiple unlabeled biomolecules simultaneously at nanomolar concentrations within minutes. Ligand-receptor binding interactions such as DNA hybridization or protein recognition occurring on microfabricated silicon cantilevers generate nanomechanical bending, which is detected optically in situ. Differential measurements including reference cantilevers on an array of eight sensors can sequence-specifically detect unlabeled DNA targets in 80-fold excess of nonmatching DNA as a background and discriminate 3 and 5 overhangs. Our experiments suggest that the nanomechanical motion originates from predominantly steric hindrance effects and depends on the concentration of DNA molecules in solution. We show that cantilever arrays can be used to investigate the thermodynamics of biomolecular interactions mechanically, and we have found that the specificity of the reaction on a cantilever is consistent with solution data. Hence cantilever arrays permit multiple binding assays in parallel and can detect femtomoles of DNA on the cantilever at a DNA concentration in solution of 75 nM.