Application of atomic force microscope in diagnosis of single cancer cells

Abstract

Changes in mechanical properties of cells are closely related to a variety of diseases. As an advanced technology on the micro/nano scale, atomic force microscopy is the most suitable tool for information acquisition of living cells in human body fluids. AFMs are able to measure and characterize the mechanical properties of cells which can be used as effective markers to distinguish between different cell types and cells in different states (benign or cancerous). Therefore, they can be employed to obtain additional information to that obtained via the traditional biochemistry methods for better identifying and diagnosing cancer cells for humans, proposing better treatment methods and prognosis, and unravelling the pathogenesis of the disease. In this report, we review the use of AFMs in cancerous tissues, organs, and cancer cells cultured in vitro to obtain cellular mechanical properties, demonstrate and summarize the results of AFMs in cancer biology, and look forward to possible future applications and the direction of development.

I. INTRODUCTION

In recent years, the number of deaths caused by cancer has increased from 7.58 × 10⁶ in 2006 to 9.6 × 10⁶ in 2018.^1,2 Despite some successful cancer treatment methods, including traditional surgical resection,^3,4 chemotherapy,^5–8 radiotherapy,^9–11 immunotherapy^12–14 and gene therapy,^15–17 cancer has been the second most common cause of death in chronic non-communicable diseases. The reason is that cancer cells can transfer in human body, proliferate and divide indefinitely, and can pretend to evade immune system attacks.

The size of human somatic cells is mostly between 10 and 20 μm. At this microscopic scale, unlike the inertial forces such as gravity at the macroscopic scale, adhesion and capillary forces between cells cannot be ignored^18–20 because the mechanical properties of cells are closely related to many diseases, such as blood diseases,^21,22 malaria,²³ and mucolipidosis type II or I-cell disease.²⁴ With regard to the acquisition of cellular information, such as cell morphology, surface roughness, adhesion, and Young's modulus, the most widely used instruments are Scanning Electron Microscope (SEM),^25–27 Transmission Electron Microscope (TEM),^28,29 and Atomic Force Microscope (AFM).^30–32 SEM is based on the interaction of electrons and matter.³³ It scans samples with a highly focused high-energy electron beam that excites various physical information. The observation of the surface topography of the test specimen is obtained by imaging, amplifying, and displaying the information.³⁴ TEM uses scatter imaging produced by accelerated and concentrated electron beams colliding into atoms in a sample.³⁵ AFM mainly relies on the interaction between atoms to obtain sample information. It converts the force of the probe into a characteristic parameter that can characterize the surface information of the sample.^36,37

AFMs are originally developed to capture cellular information, but now are also used to manipulate cells with their probe tips. AFM has already attracted widespread attention in cancer treatment from both biomedicine and engineering perspectives. Since AFM can be used in liquid phase,³⁸ nanomanipulation of cancer cells based on AFM can directly contact living cells to obtain multiple information of living cells in physiological conditions,³⁹ including physical parameters such as cell height, surface roughness, and adhesion.^40–42 The information of living cells, in particular, adhesion and Young's modulus, are believed being able to help to more accurately classify different kinds of cancer cells⁴³ and to differentiate between cancer cells and benign cells.^44,45 Furthermore, the mechanical properties of cells can be used to predict the location of metastases and can help to gain comprehensive understanding of the metastatic characteristics of cancer cells,⁴⁶ which are of great significance for the diagnosis, analysis, and treatment of cancer cells.⁴⁷

Here, we give a review of the application of atomic force microscopy in the diagnosis of single cancer cells. First, we will introduce the basic structure and basic working mode of AFM. Then, we will focus on the report of AFM in the diagnosis of single cancer cells. Finally, we will discuss the prospects of AFM and the future applications.

II. BASIC STRUCTURE AND WORKING MODES OF AFM

Atomic Force Microscope (AFM), as shown in Fig. 1,¹⁹⁸ as an important tool widely used in micrometer and nanoscale research in recent years, was successfully developed in 1986,⁴⁸ providing new ideas for microscopic research. In an atomic force microscope system, a tiny cantilever could sense the interaction between the tip and the sample being tested, and these forces could cause tiny displacements of the cantilever. When the laser is irradiated to the end of the cantilever, the displacement of the cantilever will cause the laser beam to shift, which is recorded by the laser detector (position sensitive detector) and fed back to the system, and finally presented as an image.^49,50

FIG. 1.

Basic structure diagram of AFM. Reproduced with permission from Y. Qu, Ph.D. thesis, Changchun University of Science and Technology, China, 2019. Copyright 2019 Yingmin Qu.

With the development of science and technology, existing atomic force microscopes have been equipped with the contact mode,⁵¹ non-contact mode,⁵² and tapping mode⁵³ to achieve the detection of microstructures on the surface of rigid objects such as nanomaterials.^54,55The three modes of operations of the AFM are based on the interaction between the tip and the sample.⁵⁶ When using the contact mode measurement, the probe and the sample to be tested are kept in a continuous contact state, and atomic force interaction is used to obtain information on the surface of the sample.⁵⁷ When the sample is measured using the non-contact mode, the probe is not in contact with the sample, and the sample information is obtained by the attraction of long distances between atoms.^58,59 The tapping mode works between the contact mode and the non-contact mode. The probe follows the cantilever to oscillate together at the resonant frequency, but contacts the surface of the sample periodically.^60,61 On this basis, two new modes, PeakForce tapping (PFT) mode⁶² and PeakForce Quantitative NanoMechanics (PF-QNM) mode⁶³ were derived. The PFT mode is similar to the tapping mode. The difference between the two is that the force in this mode is directly controlled by the applied force (applied maximum force) as a feedback parameter.⁶⁴ In the PF-QNM mode, the piezoelectric transducer uses high-frequency oscillation (approximately 0.25–8 kHz) to drive the engagement of the Z-axis tip, so that the tip contacts the sample surface at a speed close to 0, while the oscillation frequency of the cantilever is much lower than the resonance frequency, and achieve precise control of the press-in force.⁶⁵ A comparison of these three modes is shown in Table I.

TABLE I.

Comparison of characteristics of three working modes of AFM.

Item	Mode
	Contact mode	Non-contact mode	Tapping mode
Advantages	Fast scanning speed	No force acting on the surface of the sample	High image resolution; Does not damage the surface of soft samples
Disadvantages	Image quality will be affected by lateral forces; Soft samples may be damaged by the tip	Low horizontal resolution; Scanning speed is lower than the other two modes; Easy to cause feedback instability	Scan speed is lower than contact mode

In addition to detecting information such as nanomaterials, AFM can also detect flexible objects such as cells,^66,67 viruses,^68,69 proteins,^70,71 and even DNA molecules,^72,73 and obtain their physical information like their appearance. Due to its excellent measurement capability, such as accurate data acquisition, multi-mode selection of gas and liquid phases, and convenient operations at the micro-nano scale, AFM has often been used as an important experimental testing tool in various physical, chemical, and life science experiments^74–77 in the last few years, especially at the cell scale level.

III. SINGLE CANCER CELLS AND AFM

Together with the traditional surgical resection,^78,79 chemotherapy,^80,81 radiotherapy,^82–84 there are some emerging therapies in recent years, such as immunotherapy^85,86 and gene therapy,^87–89 focusing on specific organs using different schemes when dealing with specific types of cancers. However, these therapies have various problems. For example, they can hardly completely remove the lesions.^90–94 The chemotherapy and radiotherapy require very long period of time causing tremendous physical and psychological damages to patients and their families.^95–105 The immune therapy and the gene therapy are not very effective and have their limited scope of applications.^106–110

To overcome the deficiencies, it has been suggested that the treatments must be based on a full understanding of cancer, the most basic of which is cancer cells.^111,112 As an individual with a complete cellular structure, cancer cells have the same structure and life processes¹¹³ as ordinary cells, including cell division, differentiation, senescence, and apoptosis.^114,115

It has been found that the division of cancer cells is different from that of ordinary cells,¹¹⁶ though both rely on division to achieve the continuation of the genetic material.¹¹⁷ The division of the ordinary cells is limited by the number of cell divisions, cancer cells, on the other hand, are the only subpopulations of cells that can achieve malignant proliferation.¹¹⁸ They show almost continuously uninterrupted division under appropriate conditions. It is these abnormal divisions that make cancer a disease that is difficult to cure.¹¹⁹ In addition, cancer cells are able to move and invade other tissues and organs through blood vessels. This large transfer can occur throughout the body, which adds on further difficulty to cancer treatment.^46,120 The full understanding of the mechanism of division and movement of cancer cells¹²¹ requires multi-faceted testing of cancer cells to gather as much information as possible.

Because of subtle or large differences between individual cells (for example, between healthy cells and between healthy cells and cancer cells), single-cell level information makes it easier to describe these differences compared to the post-summary batch analysis in traditional analytical methods.

The production and development of tumors lead to the gradual loss of the biomechanical balance of cancer cells and surrounding healthy cells, resulting in significant changes in their mechanical properties.¹²² AFM has a natural advantage in detecting the changes. It precisely controls the forces exerted on cancer cells and their stromal cells, as well as the mechanical properties of the cells themselves (such as Young's modulus and adhesion), cell–matrix, and cell–cell interactions.¹²³

In recent years, AFM has been widely used in the detection of cancer cells and has made great contributions to the in-depth understanding of cancer cells. When AFM acquires cancer cell information, it acquires physical information related to cancer cells, such as cell height, cell adhesion, and Young's modulus. Changes in these single traits or multiple traits in cell characteristics can be used as a diagnostic basis for physiological and pathological conditions.^{116,124–131}

Atomic force microscope technology has three main applications in research, namely, imaging, measurement, and manipulation of samples, and some studies also use imaging and measurement functions at the same time. Whether in the process of manipulating nano-scale particles or imaging and measurement, the atomic force microscope relies on the interaction force between the probe and the sample, van der Waals force, to fulfill various requirements.

A. AFM in manipulation

When AFM is used in the manipulation of atom-level particles, the tip of an atomic force microscope can adsorb atoms, and the atomic exchange can be accomplished during the scanning process when the directional driving force generated by the tip of the atomic force microscope is greater than the threshold of interaction between the tip and the surface.^132,133 Besides that, nanomanipulation is able to manipulate atoms, molecules, and other nanoscale objects, such as placement, orientation, assembly and bending, through nano-scale operations of push-pull, shear, and pick-up.^134–136 Sugimoto et al.¹³³ first demonstrated that the near-contact atomic force microscope^56,137 can also be used for well-controlled lateral manipulation of individual atoms at room temperature and can be used as a template for nanoscale devices. Junno et al.¹³⁸ controlled a single adsorption particle with nano-precision to form a nanostructure composed of GaAs particles with a size of 30 nm on the GaAs surface and determined the true lateral size of the GaAs nanoparticles, as shown in Fig. 2.

FIG. 2.

Artificial nanostructure, the letters “nm,” made by atomic force microscope.¹³⁸ Reproduced with permission from T. Junno, K. Deppert, L. Montelius, and L. Samuelson, Appl, Phys, Lett, 66 (26), 3627–3629 (1995). Copyright 1995 AIP Publishing LLC.

However, the atomic force microscope relies on interatomic forces,¹³² and the adsorption energy of some metal adsorbents and some molecules on the insulating surface is very small,¹³⁹ which makes it difficult to manipulate atoms at normal or even at high temperature^140,141 and large insulator surfaces.¹⁴²

B. AFM in imaging and detecting

The atomic force microscope uses the weak force between the probe tip and the sample to drive the cantilever to deform the cantilever when imaging the sample. These movements and deformations are transformed into position changes that are detected by the detector, and then the structure of the sample surface information is presented at the nanometer resolution. In addition to the structure information of the sample surface, since the working principle of the atomic force microscope is based on the interaction forces between the tip and the atoms, these forces are recorded, and the characteristic physics of the sample is calculated according to the models^143–145 such as the Hertz model and the JKR model applied under different parameters, such as the Young's Modulus, that is widely used in cell biology. Therefore, the use of atomic force microscope to image and measure the sample can be achieved simultaneously on the basis of a certain model. Due to its excellent imaging capabilities and measurement precision,³⁰ AFM is widely used in cell biology research, especially in adherent cells,³² such as various cancer cell research studies in recent years.

Cancer cells used in AFM information acquisition experiments usually have two acquisition methods. One is obtained directly from the patient's tissue or body fluid, and the other is purchased from the cell bank. The difference between the two is that the characteristics of the cancer cells obtained by the first method are closer to those of the human body, and the cancer cells obtained by the second method can be continuously cultured by means of passage and the like, and are more suitable for repetitive experiments. Whether the cancer cells obtained by the two methods lead to different or even the opposite conclusion is still inconclusive.^146,147 In 2008, Cross et al.⁴⁵ isolated mesothelial cells from pleural effusions of cancer patients. After AFM testing, metastatic tumor cells were found to be 80% softer than benign cells and had 33% lower cell adhesion than normal cells. Similarly, Lekka et al.¹⁴⁸ isolated three metastatic prostate cancer cell lines (PC-3, Du145, and LNCaP) and two breast cancer cell lines (T47D and MCF-7) from the patient's organs, which were examined by atomic force microscopy, as shown in Fig. 3. It was found that the Young's modulus value of cancer cells was significantly lower than that of cells derived from benign tissues. Plodinec et al.⁴⁴ used IT-AFM to measure normal, benign, and aggressive cancerous breast tissue. In terms of stiffness distribution, both normal and benign tissues show a uniform distribution, while malignant tissue is widely distributed and has a low stiffness. After obtaining tissue from patients with cirrhosis and liver cancer, Tian et al.¹⁴⁹ discussed the lowest elastic peak (LEP) of the tissue and found that the LEP of the tumor tissue (0.42 ± 0.17 kPa) was about half of the adjacent tissues' LEP (1.10 ± 0.20 kPa). In addition, although the mechanical characteristics and tissue structure of these liver cancer tissues were diverse, they all had similar LEP (0.33–0.65 kPa). Similar conclusions were found in Ding's cervical epithelial cells obtained from patients.¹⁵⁰ By comparing the cells obtained in the inflammation group and the cancer group, it was shown that the stiffness of the cervical epithelial cells gradually increased as the severity of the cervical lesion increased. In addition, Minelli et al.¹⁵¹ found through experiments that necrotic tissue lacked rigidity and elastic structure. On the contrary, non-necrotic brain tumors (GBM and MM cell lines) are more rigid than healthy tissues. In organs that have developed cancer lesions, the volume of the lesion has a large impact on whether cancer can metastasize in the future. In prostate cancer, when the volume exceeds 4 cc, it is easy to transfer from the transition zone to the outer peripheral zone.¹⁵² Therefore, AFM has technical advantages in clinical practice, and has the potential to combine pathological analysis with surgically resected tissues.⁷⁴

FIG. 3.

The Young's modulus of the cells obtained from the (a) mammary gland (MCF7, T47D, and A184) and the cells obtained from the (b) prostate (PC -3, LNCaP, Du145, and PZHPV-7) fit Gaussian function distribution. Reproduced with permission from M. Lekka, D. Gil, K. Pogoda, J. Dulińska-Litewka, R. Jach, J. Gostek, O. Klymenko, S. Prauzner-Bechcicki, Z. Stachura, and J. Wiltowska-Zuber, Arch. Biochem. Biophys., 518 (2), 151–156 (2012). Copyright 2011 Elsevier Inc.

Since the direct acquisition of tissues and body fluids from patients involves more complicated ethical issues, most of the existing cancer cells used are derived from cell banks, and after daily culture, and relevant information is obtained. The problems encountered during the experiment and the experimental results improve the experimental and data processing methods, laying a foundation for the clinical application of AFM.^153,154 Young's modulus, which is the modulus of elasticity along the longitudinal direction, refers to the ability of cells to resist external deformation. It means that the larger the Young's modulus of the cell, the less easily the shape of the cell changes. In the experiments in which AFM is applied to cancer cell detection, Young's modulus is often focused on alone or in combination with other physical quantities (such as cell adhesion) as diagnostic criteria. At physiological temperature (37 °C), Li et al.¹⁵⁵ found that the Young's modulus of the malignant breast cancer cell line MCF-7 was significantly lower than that of its homologous non-malignant cell line MCF-10A. In addition, Calzado-Martín et al.⁴³ tested the MB-231 breast cancer cell line, further confirming that the Young's modulus of healthy cells is greater than the Young's modulus of its homologous cancer cells, while the difference between the two cancer cell lines (MCF-7 and MB-231) is not big. This conclusion was also confirmed in the non-malignant bladder cell line (HCV29) and cancer cell lines (HTB-9, HT1376 and T24).¹⁵⁶ Furthermore, Young's modulus has also been proved to be associated with cell invasiveness. Li¹⁵⁷ used AFM to detect red blood cells (RBCs) and three invasive cells (Raji, Hut, and K562), and the results (in Fig. 4) showed that invasive cancer cells were softer and easier to migrate than inert red blood cells. Similar experimental results also appeared in the ovarian cancer cell lines HEY A8 and HEY. Compared with HEY cells, HEYA8 cells had increased invasion and migration ability, while cell stiffness was reduced.¹⁵⁸ Similarly, in the relevant experiments of prostate cell line (BPH) and its homologous cancer cell line (PC-3, LNCap),¹⁵⁹ human lymphocyte lineage (normal lymphatic cancer cells and Jurkat cells),¹⁶⁰ bladder cancer cell lines (RT112, T24, and J82),¹⁶¹ and human pancreatic cancer cell lines (PaTu8988S and PaTu8988T),¹⁶² the Young's model of benign cells has been confirmed to be higher than the Young's modulus of homologous cancer cell lines, while cancer cells with high invasiveness are softer than cancer cells with less invasiveness. At the level of cell differentiation, Young's modulus can also be used as a reference. For example, well differentiated T24 cells and poorly differentiated RT4 cells have similar diameters, but well differentiated cells have a lower Young's modulus,¹⁶³ and at the same time, similar conclusions have been made in Efremov's research.¹⁶⁴

FIG. 4.

Histogram of Young's modulus for RBCs, Raji, Hut, and K562 cells. M. Li, L. Liu, N. Xi, Y. Wang, Z. Dong, X. Xiao, and W. Zhang, Sci. China Life Sci. 55 (11), 968–973 (2012), Copyright 2012 Author(s), licensed under a Creative Commons Attribution (CC BY 2.0) license.

The cells’ elastic modulus is also used as one of the indicators for measuring whether a cell is cancerous or not. Experiments have shown that malignant cells are softer than primary normal cells,^165–168 which is shown in Fig. 5, and the cellular elasticity of benign cells is more susceptible to temperature.¹⁶⁹ Meantime, cancer cells with higher metastatic potential have lower elastic modulus than metastatic cancer cells.¹⁷⁰

FIG. 5.

Elastic modulus of the central and surrounding areas of cancer cells (Hela) and healthy cells (End1/E6E7).¹⁶⁷ Reproduced with permission from K. Hayashi and M. Iwata, J. Mech. Behav. Biomed. Mater. 49, 105–111 (2015). Copyright 2015 Elsevier Ltd.

In addition to the physical parameters that characterize cell elasticity, in some studies, adhesion is also used as a criterion for discriminating the cancer cell grade and cancer cell invasion. Experiments have shown that the adhesion between high-grade cancer cells is significantly lower than that between low-grade cancer cells,¹⁷¹ and the interaction between cancer cells and endothelial cells is positively correlated with the aggressiveness of cancer cells,¹⁷² as shown in Fig. 6.

FIG. 6.

Schematic diagram of interaction between three bladder cancer cell lines (T24, J82, and RT112) and endothelial cells.¹⁷² V. M. Laurent, A. Duperray, V. S. Rajan, and C. Verdier, PLoS One 9 (5), e98034 (2014). Copyright 2014 Author(s) licensed under a Creative Commons Attribution (CC BY 4.0) license.

In addition to a single parameter, a combination of two physical quantities is often used to characterize cancer cells. Ketene's research targeted mouse ovarian surface epithelial cells (MOSEs). The results showed that the elastic modulus of advanced MOSE cells was significantly lower than that of the early counterparts, and the apparent cell viscosity also decreased significantly.¹⁷³ But not in all cases, the degree of malignancy of cancer cells is positively correlated with cell elasticity and apparent viscosity. In human chondrosarcoma cell lines (JJ012, FS090, and 105KC), JJ012 cells have the lowest modulus of the cell line tested, while FS090 typically has the highest modulus. At this time, the apparent viscosity changes as the degree of malignancy of the cancer cells increases, as shown in Fig. 7.¹⁷⁴ The three human prostate cancer cell lines studied by Bastatas et al.¹⁷⁵ showed that the elastic moduli of the two high metastatic cell lines (CL-1 and CL-2) were higher than those of the low metastatic cell line LNCaP, and the results of adhesion were also the same as that, which might mean that the enhancement of adhesion was conducive to the invasion of malignant cells and the establishment of additional blood vessels. However, Omidvar et al.¹⁷⁶ had different conclusions in the study of breast cancer cell lines (MCF-7, T47D, and MDA-MB-231). The results showed that the adhesion between cells was opposite to that of invasion, and the Young's modulus was positively correlated with adhesion. Comparably, Rebelo et al.⁴⁷ also had the same conclusions for experiments with renal cancer cell lines (A-498, ACHN) and non-tumorigenic cell lines (RC-124). Therefore, for cancer cells derived from different organs, the mechanical properties and metastatic potential may not be the same, but the correlation between mechanical characteristics and metastatic potential is unique.¹⁷⁷

FIG. 7.

Biomechanical properties (elastic modulus and apparent viscosity) of JJ012, FS090, and 105KC chondrosarcoma cell lines cultured for 2 h (a) and 2 days (b).¹⁷⁴ Reproduced with permission from E. M. Darling, S. Zauscher, J. A. Block, and F. Guilak, Biophys. J. 92 (5), 1784–1791 (2007). Copyright 2007 The Biophysical Society.

Young's modulus can be obtained by force curve fitting and calculation, and the force curve can also be used as a physical quantity to independently discuss cancer cell features in some experiments. Fuhrmann et al.¹⁷⁸ found that in cell experiments (ECP2, CP-A, and CP-D), the force-indentation curve showed a non-monotonic discontinuity similar to a jagged line, and the incidence of such events was related to whether the cells were normally development. Meanwhile, dysplastic CP-D cells had the lowest Young's modulus.

In addition, based on the measurement mode and measurement results of the atomic force microscope, there are some custom parameters like elastic shear platform moduli ($G_N^0$), glass transition frequency ($f_{\mathrm{T}}$), and loss tangent (G″/G′) as indicators for evaluating the invasiveness of cancer cells, such as the study of Abidine et al.¹⁷⁹ in bladder cell lines (experiments in RT112, T24, and J82) demonstrated a decrease in both invasive the cell elastic platform modulus and the transition frequency. The experiments of Rother et al.¹⁸⁰ in breast cell lines (MCF-10A and MDA-MB-231) demonstrated that G″/G′ increased with increasing malignancy, as shown in Fig. 8.

FIG. 8.

(a) Images of MCF-10A, MCF-7 and MDA-MB-231 cell lines obtained using AFM measurement. (b) The median values of storage modulus G′ (filled symbol) and loss modulus G″ (hollow symbol) as a function of oscillation frequency (complex shear modulus data is fitted using a power-law structure damping model). From J. Rother, H. Nöding, I. Mey and A. Janshoff, Open biology 4 (5), 140046 (2014). Copyright 2014 Author(s), licensed under a Creative Commons Attribution (CC BY 3.0) license.

However, because it needs to contact or be close to the cells during the measurement, it is impossible to obtain the characteristic quantities of suspended cells (such as the H22 cell line¹⁸¹ and the K562 cell line¹⁸²). Therefore, the detection of some blood-like cancer cells is difficult, so it is necessary to use a special technique, such as electrospinning^183,184 (shown in Fig. 9), to capture and fix cells in a liquid, after which they could be measured using an AFM.

FIG. 9.

(a)–(f) Pictures of cell characteristics within 9 days of culture. (a)–(c) are control cells. (d)–(f) were electrospun cells. Among them, the culture time of cells in [(a) and (d)], [(b) and (e)], and [(c) and (f)] was, respectively, 55 h, 80 h, and 6 days.¹⁸³ Reproduced with permission from A. Townsend-Nicholson and S. N. Jayasinghe, Biomacromolecules 7 (12), 3364–3369 (2006). Copyright 2006 American Chemical Society.

IV. SUMMARY AND PROSPECTS

AFM's unique advantages including imaging in liquid phase conditions, direct contacting with cells, and precise adjustment of applied forces, make it widely used in life sciences. High-resolution imaging of living cells in the physiological environment of the human body and further in-depth research and analysis have become hot spots and challenging topics. The complexity of the liquid environment, including solution perturbations, the complexity of living cells themselves, and the interaction between cells and probes, results in the inability to obtain high-precision images,⁷⁴ and also leads to the inaccuracy of the results obtained by the processing and analysis of cellular information. In addition, in order to more accurately describe the characteristics of cells, a large number of repeated experiments are usually carried out, which brings a huge amount of information to be processed, and increases the time for information processing and analysis.¹⁸⁵ The mechanical properties of cells, to a certain extent, can serve as markers for human health and diseases. The use of AFM to determine the mechanical properties of a single cancer cell can provide a new way for cancer diagnosis to achieve better cancer treatment. The occurrence and development of tumors is accompanied by a gradual loss of biomechanical homeostasis, leading to significant changes in the mechanical properties of cancer and surrounding cells, which can be well monitored by AFM.¹²² In the process of carcinogenesis, in order to facilitate the transfer and the establishment of blood vessels, the characteristics of Young's modulus (increased softness) and decreased adhesion are accompanied. In terms of invasiveness, more aggressive cells usually have lower Young's modulus and adhesion, but may have different conclusions in different organs and cancer cells derived from different organs. These changes may be due to the collagen content and cytoskeleton that the cells suddenly change during the process of carcinogenesis.^165,186 It is worth noting that some studies have shown that cells with higher motility are softer (usually cancer cells),^75,76 but in some research, cells with increased mobility have higher Young's moduli.^187,188 Therefore, stiffness is not necessarily the standard for identifying cells and cancer cells, but also the criteria for identifying different benign cells and different cancer cells, which are mainly reflected in the migrating ability of cells.¹⁴⁷

AFM can be used as a precise measuring instrument when used alone, and it can fully exploit the potential of AFM when combined with other technologies. At this time, multiple complementary information can be acquired at the same time, so that the physiological activities of cells can be understood from many aspects. For example, by combining AFM and confocal fluorescence microscopy, information on the mechanical properties of cells and tissues and fluorescence images can be obtained simultaneously.¹⁸⁹ Because AFM can detect changes in the mechanical properties of cells, it can be used to observe the difference in cells before and after drug action, and play an important role in drug toxicity testing, drug development, and drug screening.^190,191

In addition to cell mechanics, the resting potential and dynamic potential of the cell are also important factors to measure the cell state. Through the modification of AFM,¹⁹² or in combination with Scanning Probe Microscope (SPM)¹⁹³ and Scanning Ion Conductance Microscope (SICM),¹⁹⁴ the measurement of cell surface charge can be achieved. Besides oncology, AFM can also be used to study the vascular-related diseases, diabetic complications, kidney diseases, cataracts, Alzheimer's diseases,^195,196 and cardiomyopathies,¹⁹⁷ even applying AFM to the clinical surgery for the rehabilitation and prognosis of more diseases, to bring the gospel to human health.

DATA AVAILABILITY

Data sharing is not applicable to this article as no new data were created or analyzed in this study.