PeakForce Tapping resolves individual microvilli on living cells

Abstract

Microvilli are a common structure found on epithelial cells that increase the apical surface thus enhancing the transmembrane transport capacity and also serve as one of the cell's mechanosensors. These structures are composed of microfilaments and cytoplasm, covered by plasma membrane. Epithelial cell function is usually coupled to the density of microvilli and its individual size illustrated by diseases, in which microvilli degradation causes malabsorption and diarrhea. Atomic force microscopy (AFM) has been widely used to study the topography and morphology of living cells. Visualizing soft and flexible structures such as microvilli on the apical surface of a live cell has been very challenging because the native microvilli structures are displaced and deformed by the interaction with the probe. PeakForce Tapping® is an AFM imaging mode, which allows reducing tip–sample interactions in time (microseconds) and controlling force in the low pico‐Newton range. Data acquisition of this mode was optimized by using a newly developed PeakForce QNM‐Live Cell probe, having a short cantilever with a 17‐µm‐long tip that minimizes hydrodynamic effects between the cantilever and the sample surface. In this paper, we have demonstrated for the first time the visualization of the microvilli on living kidney cells with AFM using PeakForce Tapping. The structures observed display a force dependence representing either the whole microvilli or just the tips of the microvilli layer. Together, PeakForce Tapping allows force control in the low pico‐Newton range and enables the visualization of very soft and flexible structures on living cells under physiological conditions. © 2015 The Authors Journal of Molecular Recognition Published by John Wiley & Sons Ltd.

Nanoscale imaging and force probing of biomolecular systems using atomic force microscopy: from single molecules to living cells

“…Current high spatiotemporal resolution imaging is commonly obtained on microbial cells which have a stiff and well-defined cell surface 43,47 and animal adherent cells which can tightly adhere and spread on a substrate. 52,58,60 The dynamics of fine structures or even single molecules 194 can be captured on these cell surfaces. For animal suspended cells which do not spread on a substrate and are very soft, the imaging resolution is limited.…”

“…Studies have shown that using a long AFM tip probe whose cantilever was far away from the cell surface can effectively improve the spatial resolution of AFM images. 52 Besides, shortening the length of he cantilever can reduce viscous damping, and removing reflective gold layers from the cantilevers can reduce thermal drift and increase force sensitivity, 152,157 leading to the smaller response times of the AFM probe. Hence, fabricating adequate AFM probes can potentially benefit the improvement of AFM imaging resolution.…”

“…When applied on living cells, these forces can easily deform the soft and flexible cell surface, making it difficult to observe the fine structures (e.g., microvilli) on the surface of living cells. In 2016, Schillers et al 52 revealed the individual microvilli of living adherent cells by a new imaging mode which is called peak force tapping (PFT), as shown in Fig. 1(c).…”

Atomic force microscopy studies on cellular elastic and viscoelastic properties

“…In the past decade, wide applications of atomic force microscopy (AFM) in life science research have yielded much new knowledge about the physiological activities of individual cells. AFM can not only image the ultra-microstructures of single living cells, such as the individual *Corresponding authors (Lianqing Liu, email: lqliu@sia.cn; Ning Xi, email: xining@hku.hk) microvilli on cell surface (Schillers et al, 2016), but also can quantitatively measure the mechanical properties of single cells (Liang et al, 2016). By applying AFM indenting technique, the mechanical properties of molecules , cells (Cross et al, 2007), and tissues can be measured to improve our understanding of biological systems from the perspective of biomechanics.…”

Atomic Force Microscopy in Characterizing Cell Mechanics for Biomedical Applications: A Review

“…AFM has nanometer resolution (the spatial resolution of AFM imaging on mammalian cells is about 50 nm [23]) and can work in aqueous conditions (e.g., cell culture medium), which makes it particularly suited for detecting biological samples. With AFM, the individual microvilli on living mammalian cell surface can be visualized [24], nanoscale organizations on living bacterial cell surface can be revealed at single-molecule level [25], and the dynamic activities of single molecules at work on cell surface can be filmed by highspeed AFM [26]. Beyond topography imaging, AFM also provides high-resolution maps of cellular mechanical properties, thus acting as a reliable tool for correlating mechanics with the structures and functions of the sub-cellular structures (e.g., cytoskeleton and organelles [27]).…”