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Correspondence: John T. Connelly, Centre for Cell Biology and Cutaneous Research, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, 4 Newark Street, London E1 2AT, United Kingdom.
School of Engineering and Materials Science, Queen Mary University of London, London, United KingdomSerra-Hunter Program, Biophysics and Bioengineering Unit, Department of Biomedicine, Faculty of Medicine and Health Sciences, University of Barcelona, Barcelona, Spain
Atomic force microscopy (AFM) is a powerful technique for nanoscale imaging and mechanical analysis of biological specimens. It is based on the highly sensitive detection of forces and displacement of a sharp-tipped cantilever as it scans the surface of an object. Because it requires minimal sample processing and preparation, AFM is particularly advantageous for the analysis of cells and tissues in their near-native state. Moreover, recent advances in Bio-AFM systems and the combination with light microscopy imaging have greatly enhanced the application of AFM in biological research. In the field of dermatology, the method has led to important insights into our understanding of the biomechanics of normal healthy skin and the pathogenesis of a variety of skin diseases. In this Research Techniques Made Simple article, we review the fundamental principles of AFM, how AFM can be applied to the analysis of cell and tissue mechanics, and recent applications of AFM in skin science and dermatology.
As the primary barrier between our bodies and the external environment, the skin provides essential protection from physical injury, chemicals, radiation, and pathogens. The strength and resilience of the skin tissue are crucial for this protective function, and its biomechanical properties arise from a complex interplay between the cellular and molecular components. Whereas extracellular matrix proteins, such as collagen and elastic fibers, confer strength and flexibility to the dermis, differentiated keratinocytes (KCs) in the epidermis create a tough barrier in the outermost layer of the skin (
). Cellular mechanosensing within the skin also plays important roles in tissue homeostasis and repair through the regulation of fundamental biological processes such as growth, migration, and differentiation, and these responses are mediated by the cell’s mechanical properties and mechanotransduction machinery (
). Thus, the precise measurement and analysis of skin biomechanics are not only important for understanding normal tissue function but also for dissecting the mechanisms of disease pathogenesis.
To date, a wide range of mechanical testing methods has been developed for assessing the skin’s mechanical properties. At the whole-tissue level, tensile testing and in situ suction tests, for example, can provide useful information on the overall mechanical properties and function of the skin. However, they lack the resolution to precisely analyze the mechanical properties of specific compartments or cellular niches. Given the hierarchical and heterogeneous composition of the skin, detailed biomechanical information is therefore important for uncovering the true structure‒function relationships at the cellular and molecular level. Atomic force microscopy (AFM) is a form of scanning probe microscopy that is capable of measuring structural and mechanical properties at nanoscale resolution and is compatible with a wide range of biological samples. In recent years, it has become a key tool for analyzing cell and tissue mechanics, and AFM systems specifically designed for biological applications (i.e., Bio-AFM) have opened up new opportunities for biologists to make use of this powerful technique. In this article, we will describe the basic principles of performing AFM measurements and data analysis. In addition, we will highlight recent advances in AFM-based methods for biomechanical analyses and how these tools can be applied to dermatology and skin research.
Atomic force microscopy (AFM) provides nanoscale imaging of surface topography for biological samples in their near-native state.
Indentation testing using AFM can be used to calculate the stiffness (Young’s elastic modulus), viscosity, and adhesion forces of cells and tissues.
A combination of AFM and light microscopy can provide further insight into the relationship between specific biomolecules and biomechanics.
Limitations include the need for specialized AFM equipment and a relatively low throughput compared with those of other imaging modalities.
Principles of AFM
The core component of AFM is a sharp-tipped cantilever, which is used to scan the surface of an object in the x‒y plane and image the nanoscale surface topography (
). Deflection or bending of the cantilever is detected by shining a laser onto the back of the cantilever and measuring the reflection with a sensitive four-quadrant photodetector (Figure 1a). During an imaging experiment, the cantilever oscillates at a defined frequency, and molecular interactions between the tip and the object’s surface interfere with the oscillation frequency, which is detected by the laser. Feedback from the photodetector to the piezoelectric stage precisely controls the height of the cantilever tip in the z-direction to maintain the set oscillation frequency and record the height of the sample. In this manner, AFM can provide detailed information about a sample’s surface topography or roughness with nanoscale resolution. This imaging approach is often referred to as tapping mode (intermittent contact) and is the most common method, but it is worth noting that imaging can also be performed in full contact or in noncontact modes. Importantly, AFM imaging requires minimal sample preparation or manipulation and is less prone to processing artifacts compared with electron microscopy while maintaining similar nanometer-scale resolution. This property is particularly advantageous for the analysis of biological samples in their native state, and the current Bio-AFM systems (Table 1) include temperature-controlled liquid imaging chambers for analysis of living cells under physiological conditions.
Table 1Summary of Bio-AFM Suppliers and Current Models
In addition to imaging, AFM systems are also used to apply controlled forces onto a sample, thus acting as nanoindenters to measure the mechanics of soft cells or tissues. To estimate mechanical properties, the cantilever is slowly ramped toward the sample and then away from the sample while the deflection of the cantilever is recorded (
). By knowing the stiffness of the cantilever, the force applied can be calculated from the deflection measurements, and a force‒displacement (F-δ) curve can be generated to determine the key mechanical properties. As illustrated in Figure 1b, the shape of the approach curve is related to the stiffness (resistance to deformation) of the sample, often measured using the Young’s modulus. In addition, the area between the approach and withdrawal curves (also known as hysteresis) reflects the sample’s viscosity, and the negative force values at the end of the withdrawal curve indicate the adhesion between the tip and the object’s surface. The newest AFM systems with high speed, multiparametric imaging modes (e.g., QI or PeakForce) combine both topography imaging and mechanical measurements, rapidly recording two-dimensional grids of F-δ curves with a good spatial resolution (
). These AFM systems thus measure both topographical and mechanical information simultaneously using a single routine, yielding multiparametric maps of the same cell (Figure 2). The most recent implementations of multiparametric imaging are able to scan a 32 × 32 pixel region of a cell in less than a minute.
Mechanical parameters are obtained from the force‒distance curves by fitting the region of the curve where the tip is indenting the sample using a specific contact mechanics model (
). This is an analytical or numerical expression that predicts the deformation of a material by an indenter of known shape, under a given load, and on the basis of the material’s mechanical and morphological features. Recent demand for multiparametric imaging operations has paralleled efforts to develop contact mechanics models better suited to the mechanical nature of living cells. Cells are viscoelastic, heterogeneous along their depth, anisotropic along their area, and typically cultured over very stiff (glass) or very compliant (hydrogels) substrates. A variety of models have been proposed to tackle these mechanical features, and current models compute the Young’s moduli by correcting for the stiff or compliant substrate effects, cell viscosity, depth-dependent Young’s moduli, or cell adhesion (
). Other essential considerations for AFM studies are the selection of the appropriate cantilever stiffness, geometry, indentation depth and speed, and environmental conditions. For a more in-depth discussion of these parameters, we refer you to a previous review (
Finally, multiparametric imaging modes amass thousands of force-indentation curves for each mapped cell, and maintenance of physiological conditions allows for measuring tens of cells per culture dish. This abundance of data requires pipelines for automated analysis postexperimentally, starting from unbiased and computerized experiment calibrations and continuing with the optimal fitting of force‒distance curves with contact mechanics models. In this regard, open-source packages are available combining automation and model versatility for AFM data analysis (
Multimodal imaging and molecular recognition approaches
Although AFM can provide high-resolution structural and mechanical information about biological samples, on its own, it lacks molecular-level information that relates these physical properties to specific biomolecules. Greater mechanistic insight can therefore be achieved by combining AFM with additional microscopy systems that can image and correlate the complex cellular structures of the cell. A popular combination of AFM includes either epifluorescence, confocal microscopy, or super-resolution microscopy, where fluorescently labeled cellular components can be correlated to AFM topography images and force mapping. Such a combination of microscopes allows identification and measurement of physiological events of a wide range of biological systems, including membranes, single cells, and tissues. For example, AFM measurements of cell stiffening and shape changes can be directly related to dynamics of fluorescently labeled actomyosin contractility (
). AFM has recently been combined with super-resolution microscopy, and this methodology has, for example, led to new insights into the detailed structural dynamics of podosomes involved in cell invasion (
Analysis of the chemical and biological properties of cellular surfaces can also be investigated through the interactions between the AFM tip and the sample. This approach is often facilitated by coating and functionalizing the AFM tip with specific chemical groups or biological ligands. The adhesion and mechanical strength between the molecules on the tip and those on the sample’s surface can be measured from force‒distance indentation curves. Such functionalized tips have been used to detect the specific interactions of biological systems ranging from single biomolecules, such as antibodies, to whole living cells (
). However, a major challenge with this approach is the discrimination between specific and nonspecific adhesion forces. In this situation, the addition of control samples in which the specific interaction sites are blocked with antibodies or the use of mutants lacking specific interaction sites is essential for the correct interpretation of molecular interaction forces.
Applications of AFM in skin science and dermatology
In recent years, the application of AFM to dermatological research has provided important insights into normal cell and tissue mechanics within the skin as well as into the underlying mechanical defects in skin diseases where epidermal differentiation, barrier function, or cell‒cell adhesions have been altered. For example, AFM analysis of human KCs showed that cortical tension in the actin cytoskeleton regulates the movement of cells from the basal to suprabasal layer and helps to maintain tissue homeostasis (
). Complementing the in vitro cellular studies, AFM has also been employed to map the elastic moduli across the different layers of plantar and nonplantar skin in a cross-sectionally cut tissue, and the plantar skin cells of the stratum corneum, epidermis, and dermis were shown to be stiffer and less deformable (
). Furthermore, the thickness of the stratum corneum positively correlated with increased protection from stress-induced injury.
Beyond normal cell and tissue function, AFM has also provided important insights into the mechanical defects that underly a range of epidermal blistering diseases and impaired barrier conditions. For example, AFM indentation studies on mouse KCs lacking all type-I keratins have shown that keratins are the primary determinant of epidermal cell mechanics (
). Interestingly, the introduction of keratin-destabilizing mutations associated with epidermolysis bullosa simplex, such as keratin (K) 14 gene K14R125P, results in a greater reduction in KC stiffness than the total loss of keratins, highlighting the complexity of these dominant skin diseases (
). Our team has recently extended the application of AFM analysis of keratin mechanics by combining indentation experiments with time-lapse fluorescence imaging of GFP-tagged K14. In this experiment, we can directly measure the deformation of the keratin network by particle image velocimetry on indentation with the AFM tip, and this method has allowed us to quantify how the keratin cytoskeleton mechanically adapts to altered mechanical environments (
The role of keratins and specific intercellular adhesion complexes behind desmosomal pathologies has also been examined by combining AFM with molecular recognition approaches to measure adhesive properties of desmosomal cadherins such as desmoglein 3 (DSG 3) associated with the blister-forming autoimmune disease pemphigus vulgaris (
). A functionalized DSG 3‒coated AFM tip was used to measure the dimer formation at cell‒cell junctions of cells with wild-type and keratin null mouse KCs, and loss of keratins reduced the force created by DSG 3 dimer formation explaining the impaired intercellular adhesion observed in keratin null cells.
Finally, AFM has been applied to the investigation of how the mechanical properties of cornified cells correlate with the integrity and barrier function of the stratum corneum in healthy and inflamed skin of patients with atopic dermatitis (AD) (
), and this approach revealed that irrespective of the presence of FLG mutations, cells from patients with AD had lower elastic moduli, less natural moisturizing factors (NMFs), and reduced barrier function compared with those from healthy controls. In a separate study, AFM analysis of corneocytes from mice deficient in FLG or FLG-processing enzymes also displayed reduced moduli and correlated with a reduction in NMFs (
Together, the examples mentioned earlier show how AFM can be used to shed new light on the molecular and cellular mechanisms behind complex skin diseases. It also has the potential to become a diagnostic tool in assessing complex skin diseases, as has been explored in other diseases, such as breast cancer (
). Although the application of AFM in skin research to date has been limited to only a handful of groups, recent advances in Bio-AFM systems, standardization of experimental protocols, and availability of open-source modeling algorithms have significantly improved the accessibility of the technique and the ease with which it can be implemented in biomedical research laboratories. As these methods continue to progress and more advanced modalities are developed, AFM has the potential to become a standard tool in dermatological research and will push forward our understanding of the biomechanical underpinnings of skin health and disease.