The behavior and mechanical properties of cells are strongly dependent on the biochemical and biomechanical properties of their microenvironment. Thus, understanding the mechanical properties of ...cells, extracellular matrices, and biomaterials is key to understanding cell function and to develop new materials with tailored mechanical properties for tissue engineering and regenerative medicine applications. Atomic force microscopy (AFM) has emerged as an indispensable technique for measuring the mechanical properties of biomaterials and cells with high spatial resolution and force sensitivity within physiologically relevant environments and timescales in the kPa to GPa elastic modulus range. The growing interest in this field of bionanomechanics has been accompanied by an expanding array of models to describe the complexity of indentation of hierarchical biological samples. Furthermore, the integration of AFM with optical microscopy techniques has further opened the door to a wide range of mechanotransduction studies. In recent years, new multidimensional and multiharmonic AFM approaches for mapping mechanical properties have been developed, which allow the rapid determination of, for example, cell elasticity. This Progress Report provides an introduction and practical guide to making AFM‐based nanomechanical measurements of cells and surfaces for tissue engineering applications.
Atomic force microscopy is an indispensable tool for nanomechanical measurements of cells, cell microenvironments, and biomaterials. The mechanical properties of cells and their function are influenced by the elasticity of the extracellular matrix. Thus, understanding the nanomechanical properties is key for tissue engineering applications.
Piezoelectric properties of rat tail tendons, sectioned at angles of 0, 59, and 90° relative to the plane orthogonal to the major axis, were measured using piezoresponse force microscopy. The ...piezoelectric tensor at the length scale of an individual fibril was determined from angle-dependent in-plane and out-of-plane piezoelectric measurements. The longitudinal piezoelectric coefficient for individual fibrils at the nanoscale was found to be roughly an order of magnitude greater than that reported for macroscopic measurements of tendon, the low response of which stems from the presence of oppositely oriented fibrils, as confirmed here.
Conventional closed loop-Kelvin probe force microscopy (KPFM) has emerged as a powerful technique for probing electric and transport phenomena at the solid-gas interface. The extension of KPFM ...capabilities to probe electrostatic and electrochemical phenomena at the solid-liquid interface is of interest for a broad range of applications from energy storage to biological systems. However, the operation of KPFM implicitly relies on the presence of a linear lossless dielectric in the probe-sample gap, a condition which is violated for ionically-active liquids (e.g., when diffuse charge dynamics are present). Here, electrostatic and electrochemical measurements are demonstrated in ionically-active (polar isopropanol, milli-Q water and aqueous NaCl) and ionically-inactive (non-polar decane) liquids by electrochemical force microscopy (EcFM), a multidimensional (i.e., bias- and time-resolved) spectroscopy method. In the absence of mobile charges (ambient and non-polar liquids), KPFM and EcFM are both feasible, yielding comparable contact potential difference (CPD) values. In ionically-active liquids, KPFM is not possible and EcFM can be used to measure the dynamic CPD and a rich spectrum of information pertaining to charge screening, ion diffusion, and electrochemical processes (e.g., Faradaic reactions). EcFM measurements conducted in isopropanol and milli-Q water over Au and highly ordered pyrolytic graphite electrodes demonstrate both sample- and solvent-dependent features. Finally, the feasibility of using EcFM as a local force-based mapping technique of material-dependent electrostatic and electrochemical response is investigated. The resultant high dimensional dataset is visualized using a purely statistical approach that does not require a priori physical models, allowing for qualitative mapping of electrostatic and electrochemical material properties at the solid-liquid interface.
We performed nanomachining combined with photoluminescence spectroscopy to understand the depth distribution of nitrogen-vacancy (NV) centers formed by low energy nitrogen ion irradiation of the ...diamond surface. NV− and NV0 fluorescence signals collected from the surface progressively machined by a diamond tip in an atomic force microscope (AFM) initially rise to a maximum at 5 nm depth before returning to background levels at 10 nm. This maximum corresponds to the defect depth distribution predicted by a SRIM simulation using a 2.5 keV implantation energy per nitrogen atom. Full extinguishing of implantation produced NV− and NV0 zero phonon line peaks occurred beyond 10 nm machining depth, coinciding with the end of easy surface material removal and onset of significant tip wear. The wear ratio of for NV active, ion irradiated diamond compared to the single-crystal diamond tip was surprisingly found to be 22:1. The reported results constitute the first integrated study of in-situ machining and wear characterization via optical properties of the diamond surface containing shallow formed NV centers. We discuss possible metrology applications for diamond tools used in precision manufacturing.
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The oscillatory force profile, observed in liquids due to molecular ordering at interfaces, has been extensively investigated by means of atomic force microscopy, but it remains unclear whether ...molecular ordering is present at the tip apex. Using a displacement-sensitive, low-noise atomic force microscope (AFM) operated in dynamic mode, with a tip of radius < 1 nm, we have investigated the force profile between two approaching surfaces of the same or different hydrophilic and hydrophobic character. By directly comparing different surface chemistry interactions, we have been able to elucidate whether an oscillatory force profile is due to structured water layers adjacent to the surface, the tip, or a combination of the two. We have found that an oscillatory force profile is observed when the surface is hydrophilic in nature, irrespective of whether the tip is hydrophilic or hydrophobic. When the surface is hydrophobic, an oscillatory force profile is not measured, but rather a monotonic repulsive or a short-range attractive force is observed for interactions with a hydrophilic or a hydrophobic tip, respectively. Thus, we attribute the measurement of an oscillatory force profile, in the absence of lateral confinement effects, solely to water layers adjacent to a hydrophilic surface rather than the structuring of water at the tip apex. This is the first direct evidence that solvation forces occur solely as a result of water layers adjacent to the substrate.
Alteration in the extracellular matrix (ECM) of the optic nerve head (ONH) causes lamina cribrosa (LC) fibrosis and affects the mechanical integrity of the ONH. Increased ECM tissue stiffness drives ...myofibroblast activation leading to tissue fibrosis throughout the body. Here using primary human LC cells, we investigate the effect of substrate stiffness on profibrotic changes, which might be a key molecular mechanism driving ECM remodeling of the LC in primary open-angle glaucoma (POAG) glaucoma.
Primary human LC cells from normal and age-matched POAG glaucoma donors were cultured on substrates with defined mechanical properties of 5 and 100 kPa to replicate the range of mechanical microenvironments that cells may experience in vivo. Cell morphology, spread area, actin stress fibers, vinculin-focal adhesion formation, and α-smooth muscle actin (α-SMA) signal were examined using immunofluorescence staining. The elastic modulus of cells was measured using atomic force microscopy (AFM).
Significantly greater cell spread area along with increased actin filament development, and vinculin-focal adhesion formation (number and size) were found in both normal and glaucoma LC cells cultured on stiff substrates. These changes were positively associated with elevated cell stiffness measured by AFM. Changes in spreading and cytoskeleton organization of glaucoma LC cells were significantly more pronounced than those in normal cells. The transformation to a myofibroblast-like cell phenotype was identified in both LC cells exposed to stiffer substrates, as indicated by an increased α-SMA signal and its colocalization with the actin stress fibers.
These findings demonstrated that a stiffer cell microenvironment activates a myofibroblastic transformation in human LC cells, and therefore contributes to LC remodelling and fibrosis in glaucoma.
The atomic force microscopy (AFM) allows the measurement of interactions at interfaces with nanoscale resolution. Imperfections in the shape of the tip often lead to the presence of imaging ...artifacts, such as the blurring and repetition of objects within images. In general, these artifacts can only be avoided by discarding data and replacing the probe. Under certain circumstances (e.g., rare, high-value samples, or extensive chemical/physical tip modification), such an approach is not feasible. Here, we apply a novel deblurring technique, using a Bayesian framework, to yield a reliable estimation of the real surface topography without any prior knowledge of the tip geometry (blind reconstruction). A key contribution is to leverage the significant recently successful body of work in natural image deblurring to solve this problem. We focus specifically on the double-tip effect, where two asperities 1 are present on the tip, each contributing to the image formation mechanism. Finally, we demonstrate that the proposed technique successfully removes the double-tip effect from high-resolution AFM images, which demonstrate this artifact while preserving feature resolution.
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•Three-dimensional spatial organization influences A549 cells’ shape.•Substrate stiffness influences cytoskeleton organization and cell roughness.•In 3D, cell roughness is associated ...with invasion, but not to EMT biomarkers.•In 3D, IL-8 promotes tumour progression through PI3K/Akt and MAPK/Erk1/2 pathways.
Alternative models such as three-dimensional (3D) cell cultures represent a distinct milestone towards capturing the realities of cancer biology in vitro and reduce animal experimentation in the preclinical stage of drug discovery. Significant work remains to be done to understand how substrates used in in vitro alternatives influence cancer cells phenotype and drug efficacy responses, so that to accurately link such models to specific in vivo disease scenarios.
Our study describes how the morphological, mechanical and biochemical properties of adenocarcinoma (A549) cells change in response to a 3D environment and varying substrates. Confocal Laser Scanning (LSCM), He-Ion (HIM) and Atomic Force (AFM) microscopies, supported by ELISA and Western blotting, were used. These techniques enabled us to evaluate the shape, cytoskeletal organization, roughness, stiffness and biochemical signatures of cells grown within soft 3D matrices (PuraMatrix™ and Matrigel™), and to compare them to those of cells cultured on two-dimensional glass substrates. Cell cultures are also characterized for their biological response to docetaxel, a taxane-type drug used in Non-Small-Cell Lung Cancer (NSCLC) treatment. Our results offer an advanced biophysical insight into the properties and potential application of 3D cultures of A549 cells as in vitro alternatives in lung cancer research.
Supported dipalmitoylphosphatidylcholine (DPPC) bilayers are widely used membrane systems in biophysical and biochemical studies. Previously, short-range positional and orientational order of lipid ...headgroups of supported DPPC bilayers was observed at room temperature using low deflection noise frequency modulation atomic force microcopy (FM-AFM). While this ordering was supported by X-ray diffraction studies, it conflicted with diffusion coefficient measurements of gel-phase bilayers determined from fluorescence photobleaching experiments. In this work, we have directly imaged mica-supported DPPC bilayers with submolecular resolution over scan ranges up to 146 nm using low deflection noise FM-AFM. Both orientational and positional molecular ordering were observed in the mesoscale, indicative of crystalline order. We discuss these results in relation to previous biophysical studies and propose that the mica support induces mesoscopic crystalline order of the DPPC bilayer at room temperature. This study also demonstrates the recent advance in the scan range of submolecular scale AFM imaging.
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