Atomic force microscopy: A nanobiotechnology for cellular research

Nanobiotechnology such as atomic force microscopy (AFM) has a great application in various regimes of cell biology, offering an excellent avenue to study cellular nanotopography, nanomechanics, and nanointeraction. AFM nanotopography can provide a high resolution of nano-architectures of different cells. AFM nanomechanics have shed new light on characterizing mechanical properties of cellular structures and biological materials as well as monitoring the physiopathological processes. AFM nanointeraction measurement helps the understanding of the molecular interaction forces at a nanoscale


Introduction
Nanobiotechnology, including nanotopography, nanomechanics, and nanointeraction, has made remarkable contributions to cell biology [1,2].The cellular nanotopography and nanomechanical behaviours have been considered to be of fundamental importance for the understanding of the cellular processes, such as cellular communication and interaction, inflammation, infection, tissue development/regeneration/repair, cancer biology, and the development of new medication and biotechnological application [3].During the past decades, various techniques have been introduced to measure cellular nanomechanics and nanotopography quantitatively or qualitatively, such as micropipette aspiration, microbead rheometry, optical stretcher, fluid-based deformation cytometry, magnetic bead cytometry, traction force microscopy, microrheology, whole cell stretching, and atomic force microscopy (AFM) [4,5].
Since the world's first AFM was invented in 1986 [6], it has been widely used for cellular researches due to its advantages (Table 1) [7]: (1) objective and accurate measurement: direct three-dimensional (3D) imaging of molecular and atomicscale structures of living cells without damaging the original structures; (2) measuring under real-time physiological conditions: dynamic cellular processes can be recorded in living cells in a temperature-controlled liquid environment; (3) requiring only a few cells removed from the sample to identify their nanoproperties; and (4) biophysical characteristics of cells, such as intermolecular forces, cellular elasticity, viscoelasticity, and adhesiveness can be measured at a nanolevel.
In particular, AFM has been found be to be a great nanoscopy for cellular researches, as it offers: nanoscale non-invasive imaging of cells at an atomic level in real-time [8]; nanomechanical characterization of the cell under their physiological environment, such as viscoelasticity and adhesiveness [9]; and nanoscale measurement of the interactions between cells or between cells and biomaterials [10].For example, in the field of cancer research, AFM has been successfully used for the investigation of nanotopography, elastic modulus, and viscosity.Malignant thyroid cells have been found more deformable than normal cells [11].Normal thyroid cells have a higher viscosity than malignant thyroid cells [12], and breast cancer and oral cancer cells had lower Young's modulus than normal cells [13,14].AFM tests have also demonstrated 94% sensitivity in detecting bladder cancer, vs. 20%-80% sensitivity of currently available non-invasive diagnostics on urine samples [15].

AFM basic principles
Multiple techniques for diagnostic confirmation of a carcinoma are desired.As classical diagnostic methods of cytomorphology and immunohistochemistry are compromised by morphological variation within normal, dysplastic, and cancerous cells and the extreme biochemical diversity of diseased cells, multiple techniques for diagnosis are needed [16].AFM is now a commercially available toolkit for studying biological and nanoscale entities, and allows for imaging specific surfaces at atomic scale and measuring physical quantities such as surface charges and mechanical properties [17].It can be potentially considered as a useful tool for cancer diagnosis and detection of the malignancy degree, because it provides information about the biochemical cellular content and nanomechanical properties, which would be modified by the onset and progression of pathology [13].Importantly, AFM requires only a few cells from a patient to identify cancer cells.
AFM imaging involves a prefabricated a sharp tip with a few contact areas (nm 2 ), which is attached to onto a soft flat cantilever beam to obtain topographical information with sub-nanometre resolution (Fig. 1).A laser beam is aligned on the back of the cantilever, and a position-sensitive detector (photodiode) detects the angle of the reflected beam [18].During scanning, the cantilever constantly bends in response to the sample's 3D topography and structural details.Such vibration generates the relative height data of the sample.
There are three major operating modes in AFM: contact, non-contact, and tapping.The contact mode is the fastest and most sensitive, but it may damage the specimen, as the probe is rigid and directly contacts the specimen surface.In the noncontact mode, the cantilever oscillates at its resonance frequency, but the tip does not physically contact the specimen [19].
Tapping is most commonly used in nano-imaging as it has relatively high resolution with small specimen damage.The cantilever vibration at its resonant frequency is using a piezoelectric actuator.However, it may cause some effects on the specimen [20].

Nanotopography
AFM has been widely applied in 3D imaging biological materials in real-time, in both air and liquid (native state of the biological system) environment [21].AFM has a unique capability of imaging nanometer surface structures and nanodynamics of lipids, proteins, and DNAs in liquid without a specific fluorescent dye or by fusion with a fluorescent labelling as optical microscopy [22].Cell membrane consists of a lipid bilayer that acts as a barrier between the external environment and the cell, and also contains integral membrane proteins for the uptake and export of molecules and cell-cell communication.The fluidity of the membrane depends on the lipid and protein composition and affects membrane protein function and cell properties.AFM is a powerful tool for visualizing these supramolecular organization of cell membrane [23,24].For example, AFM has been proven useful in mapping microbial cell walls and the distribution of their constituents, including peptidoglycans, teichoic acids, polysaccharides, pili, and flagella [25][26][27].High-resolution imaging of cell membranes by AFM can be used to explore cell functions and signalling events, making AFM cell topography and quantitative imaging an attractive option for diagnosis [28].
In addition to the qualitative atomic-scale imaging, AFM can also provide quantitative analysis of the images obtained, such as roughness values, surface particle size, cell size, and cell volume values.For example, a large number of spicules were observed on the surface of leukocytes in patients with leukaemia, and the cell surface roughness was significantly higher than that in normal white blood cells [29]; breast cancer cells were shown to have a rougher surface and increased cytoskeletal volume than normal cells [30,31]; colon cancer cells were found to have altered surface stiffness [32]; It's been reported that lung cancer cells have irregular microspikes and nanoclusters [33].These studies demonstrated that AFM can be used to detect changes in the morphology of cancer cells to provide an additional method for detecting phenotypic changes complementary to traditional investigative methods such as biopsy.Apart from the topographic feature of cell membranes, high-resolution AFM is also capable of visualising the individual subunits [34].For example, cell membranes receptors, such as G-protein-coupled receptors, vitronectin receptors, and prostaglandin receptors were well described by AFM [35,36].In another example, high-light to low-light growth conditions induced major ultrastructural alterations, reflecting the adaptation of Rhodospirillum photometricum of the synthesis of cell membrane constituents [37].Apart from topographic imaging, AFM can be used to quantify the cellular mechanics [38].

Nanomechanics
The mechanical properties of cells play an important role in the homeostasis of tissues such as cell growth, division, migration, and epithelial-mesenchymal transition.It is wellknown that many cellular physiological and pathological processes change the biomechanical properties of the tissues.The studies on the influence of mechanical forces on the cellular properties and their functioning constitute nowadays an emerging field called mechanobiology [9].The new developments have included studies of cytoskeleton dynamics as related to the biomechanical aspects of contraction, adhesion, locomotion, movement of organelles and vesicles through the cytoplasm, cytokinesis, establishment of the intracellular organization of the cytoplasm, establishment of cell polarity at the cellular and molecular levels, cellular deformability and injury owing to mechanical forces, the effect of mechanical forces on cellular growth, remodeling, differentiation, and gene expression, and the mechanical properties of the whole cells [39].
Common quantities in nanomechanical mapping include Young's or elastic or stretch modulus, shear or torsional modulus, Poisson's ratio, relationship between elastic quantities, interfacial stiffness, complex modulus, storage modulus, loss modulus, loss tangent, and viscosity coefficient [40].In cell biology, stiffness, adhesive, elastic modulus (Young's modulus), and viscous properties are commonly used.It is of relevance that most of the biological materials are not purely elastic but viscoelastic instead [41].AFM has proved to be a powerful tool to quantify these nanomechanical properties of biological sample with nanoscale resolution.Some of the pathophysiological changes during healthy and diseases states appeared to be highly related with the alterations of these parameters [18].For example, AFM has been used to investigate the antiinflammatory effect of licochalcone A and triptolide on rheumatoid arthritis via measuring the elastic modulus [42,43].
Changes in the adhesive force and elastic modulus have been reported in several diseases onset and progression, including sickle cell disease, osteoarthritis, diabetes, Alzheimer's disease, and periodontal disease [44].
In addition, AFM has been playing an important role in cancer research and diagnosis (Fig. 2).Cancer as a complex disease is characterized by uncontrollable proliferation of cells that evade as they progress and disrupt the organization of tissue [45].The pathogenesis and progression of cancer are accompanied by a progressive change of the cellular mechanical homeostasis that leads to a significant alteration in the mechanical properties of cancer and stromal cells [17,46].When a normal cell transforms into a cancer cell, its shape and its internal scaffolding, known as cytoskeleton, will change.Alteration of cellular structures such as the plasma membrane, cytoskeleton, and cytosol can change the cellular mechanical properties, such as deformability, motility, and cyto-adherence [47].These changes in the mechanical properties could reduce the cancer's proliferation, but increase the invasive and metastatic potential, the migration of the cancer cells, and the cancer tissue development [48,49].In the recent decade, more studies have focused on deciphering cancer's cellular and molecular mechanism and identifying biomolecular markers to help to risk-stratify cancer and predict local invasion and metastasis.Traditionally, the cancer diagnosis is based on cytomorphology and immunohistochemistry, however, which are difficult to carry out due to morphological variation within normal, dysplastic, and cancerous cells, as well as the extreme biochemical diversity of diseased cells; the use of multiple techniques for diagnostic confirmation is desired [16].
The mechanical properties of cells, as determined by AFM, are thus novel biomarkers that could be used to discriminate cancer cells from their normal counterparts [50].During cancer development, alteration of cellular structures such as the plasma membrane, cytoskeleton, and cytosol can change the cellular mechanical properties such as deformability, motility, and cyto-adherence [47].For example, in breast cancer, cervical cancer, and oral cancer, the stiffness of breast, cervical and oral mucosal cells decreased significantly as the disease progressed [14,51,52].Recent studies of cancer have focused on the elastic properties of tumour cells [53].Living cells are viscoelastic, and the Young's modulus of the cells is affected by the loading rate of indention due to internal frictional interactions of the cell's contents and organelles [54,55].Study using AFM has revealed that viscoelastic property is one of the important markers that could be used to differentiate cancer cells from their counterparts [56].Understanding cancer from a biomechanics perspective can provide us with an alternative approach to evaluate the onset or progression of the disease.Such information could later be translated into ways to develop novel diagnostic biomarkers for early detection of cancer and to perform anti-cancer drug efficacy screening [57].
AFM-based single-molecule force spectroscopy is a great technique for measuring molecular nanointeraction with high resolution [10], characterizing the nanointeractions contributing to DNA mechanics, enzyme kinetics, ligand-receptors, membrane and protein assembly, protein aggregation, and cell adhesion [58].Nanoscale resolution of this technique allows the single molecule investigations of geometry, periodicities, cross-sectional packings, elastic moduli, volumes, mass-per-length values, and flexibilities [64].In pathology, molecular nanointeraction, such as antibody and antigen, plays a key role in diseases diagnosis.For instance, AFM study revealed that pemphigusimmunoglobulin G (IgG) reduced desmoglein transinteraction, which directly contributed to pemphigus pathogenesis [65].In pharmacology, understanding drug-target nanointeraction could bring more valuable knowledge that may potentially serve as a critical parameter in personalised medication [66].
AFM adhesion force measurements could also help in understanding the medication's physical behaviour.For example,  Sample preparation Nearly physiologically relevant conditions/under air or aqueous condition [8] Thin cross-sections, dehydration, chemical fixation, and cryo-immobilization [74,75] Freezing sample, chemical fixation, and dehydration [76] Advantages High resolution, 3D topography, measurement of mechanical properties, under normal physiological condition (in ambient air or a liquid environment), no fixation, and real-time imaging [77] Powerful magnification and the provision of information regarding compound and element's surface features, shape, size, and structure [78] Topographical visualization of structures [79] Disadvantages Low scanning speed, limitation of measurement of steep walls or overhangs, and thermal drift in the images [80] Inside a vacuum chamber, non-living objectives, and black and white images [81] High-vacuum environment and non-living objectives [82] budesonide has strong cohesive forces, but exhibits weak affinity to lactose.Salbutamol shows weak cohesive forces while having strong adherence to lactose.Therefore, salbutamol/ lactose mixture would be more consistent and stable during processing when compared with the budesonide/lactose mixture [67].These quantitative measurements of AFM could provide a range of strategies for investigating living cell adhesion to the extracellular matrix, other cells or biomaterials in their native environment [68].

Conclusions
AFM is a great nanobiotechnology for the cellular researches, including nanotopography, nanomechanics, and nanointeraction.
It also has an immense potential in cancer diagnosis and new drug development.AFM allows the researcher to decode the nanoscale structure of cells surface, cellular mechanics, and molecular interactions, contributing to our knowledge of cell functions and deeper understanding of the complex interplay between physical and biological information at the nanolevel.

Figure 1
Figure 1 Principles of AFM operation.

Figure 2
Figure 2 AFM for cellular research.

Table 1
Comparison of high-resolution microscopy