Special contribution of atomic force microscopy in cell death research

: Cell death is an important life activity in individual development. Changes in morphological and mechanical properties during cell death are crucial to identify the modes of cell death. However, due to technical limitations, little is known about these characteristics. The emergence of atomic force microscopy (AFM), a nanoscale research tool that integrates imaging and mechanical measurement functions, provides new insights into our understanding of cell death. Based on a brief introduction to the structure, principle


Introduction
Cell death is a ubiquitous biological phenomenon in all living organisms and is inseparable from organ development, aging, and the removal of damaged cells [1,2].Research on the process of cell death has become a research hotspot in the fields of biology, medicine, and pharmacy.Based on functional differences, cell death can be classified into two types: programmed cell death (PCD) and non-PCD (necrosis) [3].Currently known PCDs mainly include apoptosis, necroptosis, pyroptosis, autophagy, ferroptosis, and so on.The molecular mechanisms/signaling pathways of different death modes have been extensively explored, and meanwhile, morphological and mechanical properties are also particularly important for the characterization of different death modes.Especially in the field of oncology research, alterations in the elasticity and adhesion of individual cancer cells have been recognized as a property that promotes the spread of cancer cells [2,[4][5][6].The morphological characteristics and mechanical properties of classical cell death modes such as apoptosis have been initially explored, but the morphological and mechanical properties of new cell death modes have not been fully analyzed.Atomic force microscopy (AFM) is a nanoscale research tool that integrates imaging and mechanical measurement functions [7].Compared with other traditional microscopy techniques, AFM has many advantages.For example, in terms of sample imaging, the imaging environments are diverse, and samples can be imaged under various conditions, such as air, vacuum, or liquid, including dynamic observation of living cells [8,9].In addition, the sample is not limited by its conductive properties.Moreover, AFM does not require staining, labeling, or fixing.The possibility to operate in liquid environments and at ambient temperature moved the application of AFM extends from the materials science to the biological science [10], which needs to overcome many difficulties, including the difference between material samples and biological samples.In general, the preparation of biological samples is more complex, such as the preparation of suspension cell samples, which needs to use diverse methods (such as electrostatic adsorption and microporous membrane filtration) to fix the cells to the substrate (such as Petri dishes, cover slides) [11].During imaging of certain biological samples, such as membrane proteins protruding ∼1 nm from the membrane, the AFM contact mode can provide sub-nanometer resolution (≤1 nm) for imaging individual membrane proteins [8][9][10]12,13].Meanwhile, various physical, chemical, and biological parameters can be characterized during imaging.In addition, as a multifunctional tool, AFM is one of the important tools to study the mechanical properties of cells, including the measurements of cell Young's modulus, adhesion, and viscoelasticity [14][15][16].And AFM can measure the rupture forces between molecules or particles in the piconewton (pN) range, such as analyzing the interaction force of receptor-ligand [17][18][19][20].Over the past few decades, AFM has been successfully applied to the imaging analysis of various biological systems, including nucleic acids [21], proteins [22], cells [23,24], and tissues [23,25].
At the same time, recent research advances in AFM technology have strengthened its capabilities, such as multiparametric imaging AFM (MP-AFM) [26], and high-speed AFM (HS-AFM) [27], which enables simultaneous multiparametric imaging in a short time.The extraordinary ability of AFM makes it show a very broad contribution prospect in the field of cell death.It can clearly observe the morphological changes of different cell death methods, including changes in cell size, cell height, cell surface roughness, and the formation of cell membrane pores.In addition, AFM can measure dynamic changes in mechanical properties during cell death.Based on the previous research, this article briefly introduces the basic structure, imaging principle, and working modes of AFM and reviews its contribution in the field of cell death in detail, including the study of morphological and mechanical properties of different cell death modes (Graphical abstract, the curve in the figure is a schematic and does not represent real experimental data).At the end of this article, we also expound on the research status of AFM and discuss its current challenges and future development directions for the shortcomings of AFM.[37].The main morphological manifestations were DNA breakage, cell swelling, cell membrane pore formation, and release of cell contents (Table 1).During pyroptosis, gasdermin D (GSDMD) or GSDME is activated by caspase-1/4/5/11 or caspase-3 after cleavage, oligomerization and inserts into the cell membrane to form pyroptotic pores, leading to cell swelling and rupture [38][39][40].Afterward, in 2005, the necroptosis term was first proposed by Degterev et al. [41].Its main features include cell membrane rupture, autophagosome formation, and significant organelle swelling.RIPK1, RIPK3, and MLKL are key molecules in the process of necroptosis.Researchers found that p-MLKL has the potential to create pores in the plasma membrane resulting in membrane rupture [42,43].Ferroptosis as a novel cell death mode was first proposed in 2012 by Dixon et al. [44].The morphological characteristics were cell membrane rupture, increased density, and decreased volume of the mitochondrial membrane [45] (Table 1).

The morphology and mechanisms of different cell death modes
3 Introduction to AFM structure, principle, and working modes  [7].The invention of AFM overcomes the shortcomings of STM in measuring nonconducting samples and expands the detection from conductors and semiconductors to insulators [7].
In addition, AFM has a wide range of applications compared to other microscopes, including working in liquid environments (PBS) as well as soft samples (living cells), combining topographic imaging with force spectroscopy and nanomechanics (Table 2).AFM mainly consists of the following systems: a laser system for laser generation, a head detection system consisting of a cantilever and a very sharp tip at its end, a laser detection system that receives laser reflection, and a feedback system that processes and transmits feedback signals to the piezoelectric scanner.The working principle of AFM is shown in Figure 1a.
When the tip of the probe begin to scan the sample, the weak interaction force (mechanical contact force, van der Waals force, electrostatic force, etc.) between the probe tip and the sample causes the cantilever to swing.The deformation signal is converted into a photoelectric signal and amplified, and the signal of the interaction force between Cells do not shrink but detach and swell; pores form in the cell membrane [30] Circular protrusions formed on the membrane, about 1-5 μm [30,32] atoms can be obtained.Among them, the interaction force between the tip and the sample is related to the distance between them, which can be simply divided into attractive force and repulsive force, as shown in Figure 1b.

AFM working modes
AFM provides three commonly used working modes, namely contact mode, non-contact mode, and dynamic mode (originally called tapping or oscillation mode), as shown in Figure 1c.In the contact mode, the sample and the tip are always in contact (corresponding to segments 1-2 in Figure 1b).In this mode, high-resolution images can be obtained due to the close proximity of the sample to the tip [53].But also because of this, too close distance may cause contamination and damage to the tip, and the friction force of the tip to the sample will also cause damage to the sample and affect the quality of imaging [54].
Therefore, when applying it to softer biological samples, it is necessary to adjust the force applied to the tip, generally avoiding more than 100 pN, as excessive force may lead to reversible or even irreversible deformation [55][56][57].In the non-contact mode, the microcantilever vibrates near the sample surface (corresponding to segments 3-4 in Figure 1b) [58].In this mode, the tip is not in direct contact with the sample, which makes up for the problems of sample damage and tip contamination in the contact mode.However, compared with the contact mode, the non-contact mode has a lower resolution and is unsuitable for imaging in liquid environments.In the dynamic mode, the cantilever oscillates with a larger amplitude (greater than 20 nm) near the resonance frequency during the scanning process (corresponding to segments 1-4 in Figure 1b).This mode minimizes friction and shear forces between the tip and the sample while maintaining a resolution that is essentially the same as the contact mode.In summary, the dynamic mode is suitable for objects that are more flexible and more brittle and only weakly adsorb to supports, such as DNA [59,60], proteins [61], and fibrils of Tau [62].
4 Use AFM to detect the dynamic changes of morphological and mechanical properties of different cell death modes Some caution should be taken into account to reduce the invasiveness degree toward the delicate biological cell Imaging in a vacuum environment; the specimen must be thin.
Imaging in a vacuum environment.
samples during AFM technique.The first is sample preparation.In living cells, the position change of the sample caused by the lateral force applied by the AFM probe during scanning imaging should be avoided [63].For adherent cells, because they can be naturally attached to the substrate (such as Petri dishes, cover slides), they can be directly imaged after adherent [64].The substrate surface can also be covered with a layer of polylysine to increase the adhesion effect [65].For suspension cells, appropriate fixation methods (such as electrostatic adsorption and microporous membrane filtration) are needed to adsorb them to the substrate and then image them [11].The second is the choice of probe.Probe quality and probe parameters will directly affect the final result.Because of its small contact area with the cell, the pyramidal probe can capture the details of the geometric properties of the cell surface and is often used for cell surface topography scanning [66].The micrometer-sized spherical probe is often used to measure the more holistic mechanical properties of cells because of their stable contact [66].

AFM exploring the morphological characteristics of apoptotic cells
Human understanding of cell death has gone through a long process.Prior to the advent of cell staining and microscopy, early studies of cell death largely focused on morphological observations with the naked eye [67,68].So far, morphological observation is still one of the important basis for judging different cell death modes.And different death modes show different morphological characteristics.Currently, cell morphology is mainly observed by optical microscopy (OM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM), but these methods all have certain limitations [69][70][71] (Table 2).For example, conventional OM suffers from a resolution limit of 200 nm, making it impossible to reveal the nanostructure of cells [72,73].Second, the essential advantage of AFM over TEM/ SEM is the ability to measure the biophysical properties of biological samples under physiological conditions [74].In recent years, with the development of microscopy technology, AFM has been gradually applied to the morphological observation of different cell death modes.According to the obtained 2D and 3D topological maps of cells, relevant information such as cell volume, cell height, cell membrane surface roughness, and cell membrane pore formation can be clearly collected, which provides a strong support for further understanding the morphological characteristics of specific cell death modes.AFM has been used to observe the morphological characteristics of different cell death modes, but existing studies have mainly focused on apoptosis.Previous studies have shown that reduction in cell volume is a prerequisite for the early stages of apoptosis [75][76][77].In 2005, Hessler et al. used AFM to observe early morphological changes in human oral epidermoid carcinoma cells (KB cells) apoptosis induced by staurosporine (STS) [77].The images were obtained using the contact mode by silicon nitride cantilevers MSCT-AUNM (cantilever C) at the spring constant of about 0.01 N m −1 .AFM data showed a 50% reduction in total cell volume and a 32% reduction in total cell height, and the reduction in apoptotic cell volume preceded other key hallmarks of apoptosis, such as loss of mitochondrial membrane potential [77].Researches also indicated that there was a significant change in cell volume during the initial stage of apoptosis [77].Cells are generally in the range of an AFM scan, but even the same cell type can vary by up to a few micrometers.Therefore, there are many difficulties that need to be overcome in using changes in cell volume as a marker of early apoptosis.However, it is much more convenient to observe the surface characteristics of the membrane.Moreover, the variation of cell membrane surface roughness is an indication of early apoptosis.AFM morphological imaging allows the extraction of 3D topology-related data of cells, which enables analysis of cell membrane surface roughness.Therefore, to identify apoptosis at an early stage, in 2011, Wang et al. employed AFM to observe changes in the membrane surface roughness of hydrogen peroxide (H 2 O 2 )induced apoptosis in the mouse macrophage cell line RAW264.7 [78].Results showed that the mean roughness increased between 24 and 60 nm with increasing H 2 O 2 concentration [78].It is worth noting that, although under normal circumstances, with the occurrence of apoptosis, the surface roughness of the cell membrane shows an increasing trend.However, there are some exceptions.For example, when Cai et al. used AFM to observe the apoptotic K562 cells induced by peripheral lymphocytes.The UL20B cantilever whose length, width, and thickness are 115, 30, and 3.5 μm, respectively, was chosen in the contact mode.The oscillation frequency is 255 kHz and the force constant is 0.01 N m −1 .And they found that after co-culturing these two type cells, K562 cells showed typical characteristics of apoptosis, but the cell surface roughness was significantly reduced [79].Furthermore, the formation of cell membrane pores is one of the sources of cell surface roughness, and as the pores become deeper, the cell surface becomes rougher [80].Therefore, to better describe the morphological characteristics of apoptosis, cell membrane pores are usually observed and analyzed.These studies further reveal morphological changes during apoptosis and deepen our understanding of cell death.

Advances in AFM measurement of mechanical properties during cell death
Likewise, cell mechanics can be used to describe the state of a cell, where certain changes in mechanical properties occur during cell death.AFM has been shown to be a powerful non-destructive nanotechnology that can be used to obtain dynamic processes related to cellular mechanics, such as elasticity and adhesion.Studies have shown that apoptosis may be associated with changes in cell elasticity [81].In addition, the adhesion of cell membranes plays a very important role in cell physiology and pathological processes.Hu et al. used AFM measurement and calculated by Hertz model to obtain the Young's moduli of lymphocytes in three states of resting, activated, and apoptotic, which were 11.2 ± 5.9, 19.7 ± 4.0, and 7.1 ± 4.1 kPa, respectively, indicating that the Young's modulus of apoptotic cells was significantly reduced [82].The high values of the Young's modulus of elasticity reported in this article may be due to the limitations of experimental conditions, such as force profiles measured in air, where humidity, temperature, cell dehydration, or cell drying can have a dramatic effect on the results.For example, in 2014, Jin et al. showed the damaged effect of sodium nitroprusside (SNP) on cell surface adhesion (30% reduction) and elasticity (90% reduction) at the nano level in apoptotic chondrocytes [83].In this study, the force spectrum experiment was performed at the force loading rate is 1.2 × 10 5 pN s −1 .The mechanical properties of cells have gradually become an important characteristic to distinguish healthy cells from dead cells.Meanwhile, due to the extremely high mechanical resolution of AFM, AFM single-molecule force spectroscopy technology (AFM-SMFS) developed in AFM technology has gradually become an effective tool to measure the intra-and inter-cellular interactions of biological macromolecules at the single-cell level.Future AFM holds great potential for the mechanistic measurement of different cell death modes.

Use AFM to study the relationship between cell death and cytoskeleton
In the process of studying cell mechanics, the study of the cytoskeleton is an essential part.The cytoskeleton is important for maintaining cell mechanical properties [84], and cytoskeleton remodeling plays an important role in the process of apoptosis [30,85].Importantly, it has been shown that changes in cellular elasticity are inextricably linked to the components of the cytoskeleton in addition to the intrinsic properties of the cell membrane [86].Actin filaments and microtubules, two major components of the cytoskeleton, are severely damaged in structure, organization, and function, leading to cell death [74].For example, Jin et al. studied the cytoskeleton of SNP-induced chondrocyte apoptosis, and after SNP treatment, the F-actin and α-tubulin cytoskeleton of chondrocytes reorganized and polymerized [83].In addition, other researchers have continued to explore how the composition of the cytoskeleton affects cell elasticity.Rotsch and Radmacher found drugs that depolymerized actin filaments could significantly reduce the elastic modulus of cells, while drugs that stabilized microtubules had no significant effect on cell elasticity, suggesting that the actin network primarily determines the elastic properties of living cells [87].To learn more about the effects of how microtubules and actin filaments affect cytoskeletal mechanical properties during apoptosis.Pelling et al. indicated that cell structure was highly dynamic during the early stages of apoptosis, with F-actin in actin filaments controlling the cell's initial elastic response, while microtubules appear to control the cell's viscous relaxation for extended periods [88].Due to the limitations of AFM imaging, the most common way to monitor cytoskeletal changes is to combine AFM with confocal microscopy to study the dynamic changes of actin filaments and microtubules.However, the study by Henderson et al. showed that AFM could directly image actin filaments in living cells and observe the dynamic changes of actin filaments [89].Although researchers have a preliminary understanding of the role of the cytoskeleton in maintaining the mechanical properties of cells, the relationship between the cytoskeleton and cell death remains to be further explored.

Potential of AFM to distinguish different cell death modes
With the development of AFM technology year by year, studies using AFM to observe various cell death modes have emerged in recent years [79,90,91].Meeren et al. tested the differences in morphological and mechanical properties between three different PCD modes: intrinsic and extrinsic apoptosis, necroptosis, and ferroptosis in mouse tumor cell lines (L929sAhFas cell line) [30].In morphological measurements, the resulting topographic image was obtained using the ATEC-CONT cantilever in a JPK QI ® mode using the selected AFM nanowizard 4™ (JPK GmbH Instruments/Bruker).In cell elasticity measurements, all force curves were obtained in a contact mode using a colloidal probe containing a spherical tip of 5 μm diameter (CP-qp-SCONT-BSG, force constant 0.1 N m −1 ) and using a setpoint of 2 nN at 2 µm s −1 .During intrinsic apoptosis, cells shrink and irregular structures are formed (Figure 2a, middle left).During extrinsic apoptosis, cell shrinkage was increased with concomitant apoptotic body generation (Figure 3a, middle right).During necroptosis, cells did not shrink but detached and swelled, and pores of different sizes were formed in the cell membrane (Figure 2b, middle).During ferroptosis, circular protrusions of 1-5 μm were observed to form on the cell membrane surface (Figure 2c, middle).In addition, the data showed that as the cell death process progressed, the cell surface roughness of these three cell death modes increased significantly.And the smallest increase is necroptosis.Overall, the AFM data clearly showed morphological characteristics in different cell death modes.Second, Meeren et al. also used AFM to measure the elasticity of multiple cells in these three PCDs.In the process of intrinsic apoptosis, Young's modulus decreased rapidly 15 min after induction (Figure 2a, bottom left) and remained basically stable after the decrease.In contrast, for extrinsic apoptosis, Young's modulus showed a trend of first increasing and then rapidly decreasing 60 min after induction (Figure 2a, bottom right).For both necroptosis and ferroptosis, Young's modulus showed a continuous decreasing trend (Figure 2b and c, bottom).When comparing the Young's modulus of these three cell death modes, it was found that the Young's modulus of apoptosis (intrinsic and extrinsic apoptosis) decreased more rapidly compared with necroptosis and ferroptosis.Therefore, using AFM to study different cell death modes will help us to more clearly understand the dynamic changes of morphological and mechanical properties in the process of cell death.

AFM visualizes membrane pores in different cell death
A membrane pore can be defined as any local membrane perturbation that allows passive flow of molecules [96].Different types of pores formation in the membrane may lead to different types of cell death [31].For example, after induction of apoptosis, BAX and BAK will aggregate into pores in the mitochondrial membrane, release pro-apoptotic factors, and start the apoptosis process [34].In addition, during apoptosis, perforin (PFR) released by T/NK cells can also form pores in the cell membrane [97].Similarly, the gasdermin family (such as GSDMD or GSDME), the ultimate executor of pyroptosis, induces the occurrence of pyroptosis by punching holes in the cell membrane [38].MLKL is the only known effector molecule associated with necroptosis [98].Available data suggest that p-MLKL has the potential to create pores in the plasma membrane resulting in membrane rupture [42,43].Unfortunately, we are not yet able to understand the structure and topology of MLKL in the cell membrane [99].In addition, the membrane attack complex (MAC), a terminal pathway common to the three complement activation pathways, can also be assembled by soluble monomeric proteins and form killer transmembrane pores that mediate cell death [100].AFM has successfully imaged the morphology of membrane attack complex structures on the cell membrane (Figure 3a) [92,101].Similarly, in the past experiments, SEM, TEM, and other microscopes were mostly used to observe the formed membrane pores.However, because of the harsh imaging environment, it is usually difficult to observe clear membrane pore images and the shape of the formed oligomers [102][103][104].In contrast, AFM, as a microscope that allows the acquisition of native membrane images at sub-molecular resolution, enables a clearer observation of the dynamic assembly process of transmembrane pores [105].Therefore, we broadly review relevant studies on the dynamic visualization of membrane pore formation mediated by various molecules by AFM.These studies reveal real-time morphology during pore formation and greatly advance our understanding of pore formation-mediated cell death [106][107][108].

AFM visualization of BAX/BAK and PFRmediated apoptosis pore formation
BAX and BAK are pro-apoptotic members of the BCL-2 family required for mitochondrial outer membrane permeability (MOMP) and play a key role in apoptosis [33].Under normal conditions, BAX and BAK are present in healthy cells in inactive forms.After receiving an apoptotic signal, they are activated and oligomerically inserted into the MOM, which is accompanied by an increase in MOMP, and then BAX and BAK form giant pores [33,34,90].However, the mechanism of how BAX and BAK aggregate and form apoptotic pores is still not fully understood.A recent study showed that AFM analysis of supported lipid bilayers (SLBs) prepared from proteoliposomes containing activated BAX monomers revealed that BAX formed linear, arc-shaped, and ring-shaped in SLBs of varying sizes and shapes.In other words, AFM can observe the formation of membrane pores associated with this protein (Figure 3b), which are not uniform in size and shape but are generally circular with diameters between 24 and 176 nm [93].In the same way, Cosentino et al. observed the structure formed by BAK in SLBs and compared it with the pores formed by BAX.BAK also formed straight lines, arcs, and ring structures in SLBs (Figure 3c), but the pores formed by BAK were smaller and more uniform, and the rings had an average pore radius of about 8.12 nm [94].
As a pore-forming protein, PFR can form oligomeric pores in target cell membranes, which allow the entry of pro-apoptotic granzymes, thereby causing apoptosis of target cells [109,110].Leung et al. visualized the PFR nanopore assembly in real time, where PFR first forms loosely but irreversibly bound, short prepore oligomers on target cell membranes.These short oligomers, after insertion into the membrane, recruit additional prepore oligomers, facilitating further assembly to form larger arc-and ringshaped transmembrane pores (Figure 3d) [95].

AFM visualization of gasdermin family protein-mediated pyroptosis pore formation
Gasdermin family proteins, as the final executors of pyroptosis, consist of six members in humans, namely GSDMA, GSDMB, GSDMC, GSDMD, GSDME (DFNA5), and PJVK (DFNB59) [111,112].Among them, GSDMD, as one of the earliest discovered, most widely studied, and most indepth members of the gasdermin family, can be activated and cleaved by caspase 1/4/5/11 to form N-GSDMD and C-GSDMD.N-GSDMD acts directly on the cell membrane, forming pores in the membrane and triggering pyroptosis [111][112][113].However, how the N-GSDMD domain assembles the pores and the mechanism of penetrating the plasma membrane remains poorly understood.To address this issue, Mulvihil et al. applied high-resolution and time-lapse AFM to directly image the pore-forming process of N-GSDMD on supported lipid membranes (SLMs) [91].Results showed that N-GSDMD released from GSDMD cleavage by caspase-1 can assemble into slit-, ring-and few arc-shaped oligomers (Figure 4a).And each oligomer structure occasionally forms transmembrane pores penetrating the SLMs.
The structure height of slit-and ring-shaped oligomers protruding from the SLM is about 3.6 nm, indicating that they are almost completely inserted into the membrane.The analytical data also showed that the diameters of the ring-shaped oligomers were widely distributed between 13.5 and 33.5 nm, with an average value of approximately 22.6 nm [91].These findings are consistent with the study by Sborgi et al. that N-GSDMD binds to lipid membranes and forms arc-, slit-, and ring-shaped oligomers, and the formed arcs and slits may fuse into ring-shaped N-GSDMD oligomers of variable diameter with an average value of 21 nm [103].On the basis of AFM imaging, it can be speculated that the pathway for N-GSDMD to form pores is roughly as shown in Figure 4b.Furthermore, it is necessary to explore whether the pore-forming activity of GSDMD-N is also present in other gasdermin family members.Mari et al. characterized the pore-forming activity of mouse GSDMA3 by high-resolution time-lapse AFM [102].It was found that GSDMA3 oligomers assemble on the SLMs and remain in a mobile or attached state.And once inserted into the membrane, it oligomerizes to form arc-, slit-, and ring-shaped oligomers, each of which also forms transmembrane pores, the pathway of N-GSDMA3 to form pores is shown in Figure 4b [102].In addition, Liu et al. visualized the pore-forming process of GSDME on the cell membrane by AFM [31].Overall, these studies collectively suggest that the gasdermin family proteins are the direct and ultimate executor of pyroptosis.The high-resolution information of the pyroptotic pores shows the unique value of AFM in the study of pyroptosis.

Problems with AFM visualization of membrane pores
To date, AFM has made great progress in visualizing membrane pore formation, but most studies have imaged pores on artificial liposomes or SLMs [91,93,103], and only a few studies have imaged membrane pores on real living cells [31].This could be due to the strict requirements of highresolution imaging (nanoscale) and sample preparation problems, imaging in real cell membrane pores is difficult and complicated [31].Because cell membranes are mainly composed of membrane proteins and phospholipids.Artificial lipid bilayers model (artificial membranes) can simulate cell membranes and keep their function intact [114].Therefore, the artificial membranes are good alternative to cell membranes.It provides a lot of information about the morphology and properties of a phospholipid bilayer.However, there are still some limitations in the use of artificial liposomes and SLMs for pore visualization, including the lack of a membrane protein-intrinsic cytoskeleton, leading to possible discrepancies among pores in liposomes and SLMs and the actual cell membrane, generating possible errors in observing the dynamics of pore formation [31,115].For example, related studies have shown that in real cells, PFR-induced pore size is about 200 nm, much larger than that detected from SLMs (about 10-20 nm) [95,115].In addition, the size of pores formed by GSDMD proteins in real cell membranes and SLMs is also inconsistent [31,91,103].Therefore, the results obtained using artificial liposomes and SLMs as research subjects may not reflect the relationship between cell membrane pore formation and cell death.To make a better connection between the formation of membrane pores and cell death, the study of real cell membrane pore formation using AFM should be accelerated.However, there is another problem with the visualization of membrane pores by AFM.Although studies are comparing the membrane pores depth with the cytoplasmic membrane thickness to confirm that the membrane pores imaged by AFM are real cell membrane pores and not from membrane depression or folds [31].We still do not know how to exclude that only hollows were formed as the AFM tip cannot probe the inner part of the cell nor the underneath part of a supported lipid layer.
6 Study of cell death-related molecules using AFM-SMFS The reasons for different cell death modes are not the same, it has been described above that the changes in morphological and mechanical properties of different cell death modes induced by different substances can be directly detected by AFM.However, as with other imaging techniques, we cannot obtain quantitative information on intermolecular or intramolecular interactions from AFM images, which also leads us to be unable to fully determine that cell death is induced by a specific substance.And the singlemolecule force spectroscopy technique of atomic force microscopy (AFM-SMFS) is a good solution to this problem [116].Since this technique requires tip-sample interaction, we can achieve this by functionalizing the AFM tip with particular chemical groups/ligands/antibodies. The main methods for attaching specific molecules to the AFM tip include physical adsorption, specific interaction, and chemical coupling (silanization, covalent coupling).In the experimental process, according to the nature of the sample, different connection methods can be used [117][118][119].After functionalization, AFM-SMFS allows the measurement of the adhesion and mechanical strength of specific bonds formed between the tip and individual sample molecules.Therefore, AFM can not only be used for imaging but has also become an indispensable measurement tool for studying biological macromolecular interactions.
The rapid development of AFM-SMFS has made it successfully applied to the analysis of single-molecule mechanical properties and intermolecular interactions, including DNA melting and dynamic structural changes [120], the dissociation mechanisms of DNA duplex [121], protein folding and unfolding [122], and interactions between biological macromolecules (protein-protein interaction, protein-nucleic acid interaction, protein-ligand interaction, etc.) [123][124][125].However, past studies have typically used only one biomolecule to functionalize the AFM tip when applying single-molecule force spectroscopy, but Pfreundschuh et al. further developed this technique [126].Pfreundschuh et al. functionalized the AFM tip with two different ligands to map the two binding sites of human G protein-coupled receptors [126].It can be seen that AFM-SMFS has great prospects in the field of biological research.Therefore, in recent years, AFM-SMFS has also been gradually applied in the field of cell death.pf-80 is an anti-PFR monoclonal antibody that recognizes PFR epitopes without interfering with plasma membrane binding of PFR [109,127].In studying PFR-induced apoptosis in bulk tumor cells [128] and tumor-repopulating cells (TRC, low flexibility prevents membrane pore formation caused by PFR released by cytotoxic T cells), they attached pf-80 to the AFM tip and examined the adhesion between pf-80 and PFR (Figure 5a, left) [129].The results showed that in bulk tumor cells, the adhesion between pf-80 and PFR was very high.But in TRC cells, the adhesion between pf-80 and PFR was very low, which may be due to the decreased expression of MYH9 (a motor protein that interacts with PFR) in TRC, which has the function of cross-linking and hardening F-actin.Its reduced expression may lead to a decrease in the interaction force between stable pf-80 and PFR, which ultimately leads to less adhesion between pf-80 and PFR in TRC cells [129].Epidermal growth factor receptor (EGFR) is a cell transmembrane protein whose overexpression is common in many cancers, which can be activated by binding to epidermal growth factor (EGF).In other words, low expression of EGFR can induce tumor cell death.Resveratrol, as an anti-tumor drug, can inhibit the activity of EGFR, thereby inducing tumor cell death [86,130,131].Zhang et al. used EGF-functionalized AFM probes to study the cell surface EGFR expression changes after treatment with different resveratrol concentrations (Figure 5a, right) [86].The results showed that there was no significant change in the unbinding force of the low-dose resveratrol-treated group (up to 20 μg ml −1 ) compared with the untreated group.However, when cells were treated with 40 μg ml −1 resveratrol, the unbinding force was significantly reduced.Furthermore, they also demonstrated that when EGF (100 mg ml −1 ) is added gently to untreated MCF-7 cells, the interaction forces between EGF and EGFR were specifically blocked (Figure 5b) [86].Thus, can we apply a similar idea to other cell death processes (Gasdermin family-mediated pyroptosis, MLKL protein-mediated necroptosis) using single-molecule force spectroscopy?This will help us to understand the dynamic changes and mechanisms of single molecules in the process of inducing cell death.

Discussion and further perspective
Clearly, AFM has emerged as a powerful imaging tool capable of imaging biological systems at high resolution (nanoscale).AFM topography can be used to characterize the morphological characteristics of different cell death modes.Analysis of cell biomechanical properties can detect the early onset of cell death and the dynamic changes in mechanical properties during death, thereby distinguishing different cell death modes [30].Notably, AFM is currently the only force measurement technique that can map the nanoscale lateral distribution of individual molecular recognition sites on biological surfaces, which is beyond the reach of traditional biochemical methods [118].And in recent years, great progress has been made in enhancing the function of AFM, including the development of different AFMs such as multiparametric AFM (MP-AFM) [26], molecular recognition AFM (MR-AFM) [132], multifrequency AFM (MF-AFM) [61], high-speed AFM (HS-AFM) [27], and fluidic force microscopy (FluidFM) [133].At the same time, to better image and identify the complex structure of some biological systems, AFM can also be combined with other complementary techniques, including OM, fluorescence microscopy, confocal microscopy, super-resolution microscopy, etc.The most commonly used combination of AFM is fluorescence microscopy or confocal microscopy [10].For example, in addressing the limitations of AFM imaging on the cell surface, a study combined AFM with confocal microscopy to directly penetrate the cell membrane and localize to the nucleus through the AFM tip to measure the elasticity of the nucleus [134].Obviously, this has become a hot spot of current AFM research and a trend of future AFM development.The combination and development of these technologies and functions have greatly enhanced the roles of traditional AFM, but the application of AFM still faces some challenges.
First, how to further improve the spatiotemporal resolution of AFM.At present, conventional AFM is only used to take static snapshots or very slow dynamic processes, because it takes up several minutes to acquire an image of each sample, which is far more than the time required for normal cellular physiological activities.Although highspeed atomic force microscopes with high temporal resolution in the subsecond range have been developed in recent years [135].However, due to the limitations of its high-speed imaging, this technique is currently only used for imaging small and relatively flat biological samples, such as single proteins and DNA molecules [136,137].But, for some large biological systems, the spatial resolution of AFM imaging becomes another challenge.For example, the resolution of current AFM imaging of mammalian cells is limited to 50-100 nm, which makes it impossible to observe the individual components of cell-surface machinery [132].The imaging resolution of microbial cells is slightly higher, but only about 10 nm [10].So, next, we should consider how AFM can maintain its high temporal resolution and high spatial resolution at the same time when imaging different biological samples.
Second, AFM is low throughput and labor intensive.Current AFM experiments are manual rather than automated.Meanwhile, the traditional AFM takes several minutes or even longer to process one sample, and the next one can only be processed after one sample is processed, which makes the entire operation very time-consuming and labor-intensive.In addition, when measuring the mechanical property changes during cell death based on the force-distance curve, it is usually necessary to measure and analyze thousands of force curves, which will undoubtedly further increase the time and labor consumption.In response to this problem, Chopinet et al. provide a novel modality called quantitative imaging™ (QI™), which overcomes the data processing problems associated with acquiring thousands of force curves [138].However, since this model has not yet been popularized, further researches are needed on related issues.Furthermore, the technical requirements for operators during AFM operation are very strict, especially for dynamic monitoring of living cells, which requires operators to have rich experience and good patience.Therefore, it is imminent to study the AFM of automated operation and simplified operation of data processing systems.
Finally, AFM does not need to be marked to be a double-edged sword.AFM does not need to mark the sample when observing the sample, which is one of the advantages of AFM.However, as the research system becomes more complex, this will turn into a weakness of AFM.For example, many proteins do not exhibit distinct morphologies, which are difficult to distinguish using AFM [8].Although the existing technology has been able to functionalize the AFM probe, using the tip with specific Special contribution of AFM in cell death research  13 molecular modification to specifically recognize the sample molecule.However, this technique is complicated and timeconsuming in the molecular modification process, the experimental process is difficult to standardize, and the damage to the tip is unavoidable [139].Therefore, further development of techniques that allow molecular-specific recognition by AFM is critical for imaging complex biological systems.
Generally speaking, the powerful functions exhibited by AFM make its research prospects in cell death immeasurable.However, as mentioned above, AFM still has many deficiencies in studying the physiological process of cells.Although some novel AFMs have been developed under the joint efforts of experts in multidisciplinary fields, the underlying problems remain unsolved.We expect a new and more powerful AFM that integrates multi-mode, multiparameter, multi-frequency, and high-speed modes in the future.Coupled with the advancement of various complementary technologies, this will allow us to more clearly explore the unsolved problems of the cell death process and the entire field of biology.

Figure 1 :
Figure 1: AFM.(a) Schematic diagram of AFM operation.(b) Force-distance curve by AFM.(c) Three working modes are commonly used in AFM.

Figure 3 :
Figure 3: MAC, BAX, BAK, and perforin pore morphological characteristics detected by AFM scanning.AFM imaging 3D topography of the MAC, BAK, BAX, and perforin pore (top of each image).The MAC, BAX, BAK, and perforin molecules around the pore protrude around the membrane plane, as confirmed by the height cross-sections below each image (corresponding to the gray dashed line in the AFM image) (bottom of each image).(a) Open access source, under Creative Commons license, from [92].(b) Reprinted, with permission, from [93].(c) Open access source, under Creative Commons license, from [94].(d) Reprinted, with permission, from [95].

Figure 5 :
Figure 5: The specific recognition and detection of the molecule related to cell death by single-molecule force spectroscopy technique of atomic force microscopy (AFM-SMFS).(a) Schematic diagram of measuring the interaction between perforin and its monoclonal antibody pf-80, epidermal growth factor (EGF) and its receptor EGFR on the cell membrane by AFM-SMFS technology.(b) Typical force curve of EGF and EGFR interaction with specific unbinding peaks on cancer cells.

Table 1 :
The morphological features of different cell death modes