Biomembrane force probe (BFP): Design, advancements, and recent applications to live‐cell mechanobiology

Mechanical forces play a vital role in biological processes at molecular and cellular levels, significantly impacting various diseases such as cancer, cardiovascular disease, and COVID‐19. Recent advancements in dynamic force spectroscopy (DFS) techniques have enabled the application and measurement of forces and displacements with high resolutions, providing crucial insights into the mechanical pathways underlying these diseases. Among DFS techniques, the biomembrane force probe (BFP) stands out for its ability to measure bond kinetics and cellular mechanosensing with pico‐newton and nano‐meter resolutions. Here, a comprehensive overview of the classical BFP‐DFS setup is presented and key advancements are emphasized, including the development of dual biomembrane force probe (dBFP) and fluorescence biomembrane force probe (fBFP). BFP‐DFS allows us to investigate dynamic bond behaviors on living cells and significantly enhances the understanding of specific ligand‐receptor axes mediated cell mechanosensing. The contributions of BFP‐DFS to the fields of cancer biology, thrombosis, and inflammation are delved into, exploring its potential to elucidate novel therapeutic discoveries. Furthermore, future BFP upgrades aimed at improving output and feasibility are anticipated, emphasizing its growing importance in the field of cell mechanobiology. Although BFP‐DFS remains a niche research modality, its impact on the expanding field of cell mechanobiology is immense.


INTRODUCTION
The field of mechanobiology, which explores the interplay between single cells and their mechanical environment, has witnessed significant advancements in recent years. By combining insights from biophysics and biomechanical engineering, [1] mechanobiology has greatly benefitted from dynamic force spectroscopy (DFS) techniques, such as atomic force microscopy (AFM), optical tweezers (OT), and biomembrane force probe (BFP). [2] BFP technology has enabled biomechanical analysis at both microscale (single cell) and nanoscale (single molecule) levels, providing mechanistic insights into a wide array of biological processes associated with prevalent human diseases, including cancer, thrombosis, and inflammation ( Figure 1). Recently, BFP was even used to elucidate new findings into the SARS-CoV-2 viral infection, specifically mechanical activation of the spike protein. [3] Further, DFS coupled with BFP has allowed for manipulation, characterization, and visualization of single ligand-receptor interactions and conformational changes with subsequent signaling events on live cells. [4,5] Originally developed by Evan Evans in the mid-1990s, BFP stemmed from micropipette aspiration techniques and was intended to investigate the strength of single molecular bonds under sub-microscopic forces at biological interfaces. [6] Over the past decade, BFP systems have been established by several groups, and the protocols for setup and operation have been reviewed alongside other nanotools. [2,[7][8][9] The conventional BFP setup is represented in Figure 1 and consists of two opposing micropipettes aligned along their horizontal axis' (left: 'Probe'; right: 'Target'). While the in-house setups may vary slightly, the basic structure of each BFP is similar. The left micropipette, which is held stationary, aspirates a human erythrocyte, or red blood cell (RBC), which is biotinylated. A streptavidin-coated glass microbead, which can be coated with ligands of interest, is attached on the apex of the RBC (Figure 1). On the right, another micropipette, controlled by a piezoelectric translator holds the opposing bead or cell bearing complimentary receptors and is driven to impinge the probe bead in a repeated "approach-push-retract-holdreturn" test cycle. [10] A third micropipette (termed 'Helper') is typically utilized to attach the streptavidin-coated glass bead onto the RBC apex. Micropipette aspiration applies a pressure that allows the RBC to serve as a hypersensitive force transducer. Typically, the BFP is configured with at least two cameras, an inverted microscope with a dry objective lens (e.g., 40X/NA0.75), a mercury lamp as a light source, and several video tubes. One camera operates at high speed to track the displacement of the RBC-Probe edge, while the other allows real-time visualization of the ongoing experiment. [11] Enabled by fast video processing, the RBC-Probe edge is tracked along the pulling direction at a video rate of up to 1600 fps provided the images are reduced to a 24-30-line strip across the bead. [12] Over the past 20 years, BFP has been utilized as a dynamic force spectroscopy (DFS) technique to continuously contribute to the field of mechanobiology by providing high resolution in position, time, and force (2-5 nm, 0.3-0.5 ms, and 0.2-0.5 pN). [13] Aside from the conventional BFP, advancements in the setup have led to the development of both a dual BFP and fluorescence BFP setup. The BFP-DFS applications include ligand binding kinetics, [14][15][16][17][18][19] cytoskeleton mediated receptor activation, [20][21][22][23][24] receptor conformational changes, [25,26] and dual receptors crosstalk, [27,28] which have significantly contributed to research in cancer biology, thrombosis, and inflammation.
As an ultrasensitive DFS technique, BFP has expanded in scope and application over the past decades. In this review, we first introduce the conventional BFP and delve into updated techniques using this technology. We then underscore BFP's versatility by summarizing key upgrades and their impact on the field of mechanobiology. Furthermore, we examine the novel clinical contributions facilitated by this technique in  of  F I G U R E  BFP dynamic force spectroscopy workflow. This flow chart outlines the key steps in the biomembrane force probe (BFP) workflow, starting with (1) reagent preparation, followed by (2) BFP assembly, and (3) BFP test cycles execution. (4) The test cycles comprise four BFP assays and dynamic force spectroscopy (DFS) modes: adhesion frequency assay, thermal fluctuation assay, force clamp assay, and force ramp assay. (5) Data generated from these assays are analyzed. The BFP test cycle (3) illustrates the red blood cell (RBC) deflection and the positions of the probe and target beads during a touch cycle. The target bead approaches and impinges the probe bead, then retracts. The vertical dashed lines indicate the zero-force position of the RBC apex. Key variables involved in the BFP experiment are also depicted: k RBC (spring constant of the RBC), Δp (aspiration pressure applied to the probe pipette), R p (pipette radius), R 0 (RBC radius outside the pipette), R c (radius of the contact area between the RBC and the bead), R B (probe bead radius), and Δx (displacement of the probe bead and the deformation of the RBC). The radius of the RBC tail, l, should be comparable to R 0 . The variables k RBC and the binding force, F, can be quantified using these variables. the context of cancer biology, thrombosis, and inflammation. Finally, we discuss potential developments and future implications in the field.

 CONVENTIONAL BFP SETUPS AND PRINCIPLES
Initially, the conventional BFP was developed to measure the strength of single ligand-receptor bonds and receptormembrane anchoring with high force, spatial, and temporal resolution (≈1 pN, ≈3 nm, and ≈0.5 ms), [38] over a wide range of loading rates (10 −1 to 10 5 pN s −1 ). [39] The brilliance of BFP lies in its ability to test the strength of bonds as the maximum force of a molecular attachment upon bond failure. As described earlier, conventional BFP uses a pre-swollen RBC (a naturally elastic biomaterial) as an ultrasensitive force transducer with a spring constant range from 0.1-3 pN nm −1 [38] (Figure 2). Central to the BFP methodology is the determination of the spring constant, of which there have been several approximations suggested. [40][41][42] Evans et al. first studied the deformation of an RBC submitted to a force in 1995 and defined the erythrocyte behavior at small deformation as a Hookean spring with a constant (k RBC ) where Δp is the aspiration pressure at probe pipette tip, R 0 , R p , and R c are the radii of the RBC, the probe micropipette inner orifice, and the circular contact area between the probe bead and the RBC, respectively [9] as demonstrated in Figure 2. As benchmarked and summarized by Ju and Zhu, [43] Evans' model is the typically used when the tail of the RBC (l) aspirated into the micropipette is comparable in length to the inner radius of the micropipette (R p ) such that l ≈R p . Further, Simson et al. redefined the RBC spring constant (k RBC ) as [41] Like the model proposed by Evans' a few years prior, Simson's model provides an analytical expression for the stiffness of the transducer (RBC) provided only small elongations are submitted to the RBC (Δx < 200 nm). [41] As reported by Ju and Zhu, Simson's model functions well when the length tail of the RBC (l) is slightly greater than the inner radius of the micropipette (R p ) such that l > R p , provided the radius of the micropipette does not exceed 1 μm. [43] Finally, Heinrich et al. further corrected the expression for the BFP spring constant (k RBC ) as such [40] In contrast to the previous models, Heinrich's model predicts a spring constant (k RBC ) that is significantly larger [40] . Once again, Ju and Zhu found that Heinrich's model is preferred in cases where the aspirated RBC tail (l) is significantly longer in length than the inner radius of the micropipette (R p ) such that l >> R p . [43] Regardless of the model used for spring constant approximation, several assumptions are made. Specifically, the assumption that axial symmetry is preserved along the center line of the micropipette. As such, upon BFP assembly, the probe bead must be placed very carefully on the apex of the RBC in order to ensure that all external forces acting on the bead act exclusively along this axis of symmetry. Additionally, in all scenarios, the RBC membrane in contact with the bead (R c ) is assumed to be fixed. Practically, this reaffirms the critical importance of ensuring that the probe bead is securely attached to the RBC apex during an experiment. Notably, biotinylation of the RBCs needs to be well controlled and monitored to allow for optimal bead attachment. [44] Detailed protocols for RBC biotinylation, bead silanization, and bead functionalization have been published previously. [44,45] Regardless of the spring constant approximation used, force (F) is calculated using Hooke's law, where k RBC is the spring constant of the RBC and Δx is the displacement of the probe bead, which is tracked by a valley detection algorithm. [27] The target micropipette is then retracted and held with a clamped force while RBC is deformed by the ligand-receptor bond forces whose displacement is monitored in real-time by fast video imaging. Table 1 summarizes the key mechanical parameters that can be manipulated with the conventional BFP set up-contact time (s), impingement force (pN), and ramping rate (pN s −1 ). By analyzing the BFP force versus time traces through the entire approaching, impinging, retracting, clamping, and bond dissociating test cycle, bond 2D kinetics (association k on and dissociation k off ), [32,46,47] bond lifetime, [24,48,49] molecular stiffness, [50] and intracellular events (with the aid of fluorescence microscopy) [25,51,52] can be obtained (Table 3).

. Adhesion assays
In this section, we highlight key BFP assays and DFS modes, starting with the adhesion frequency assay. Originally developed for use in the context of micropipette aspiration, where a human RBC serves as an adhesion sensor, this method can be modified and applied to BFP. The micropipette adhesion assay was developed by Chesla et al. to measure two-dimensional ligand-receptor binding kinetics, based on the premise that adhesion probability depends on contact time and ligand and receptor densities. [53] Since its development, the assay has been validated using selectins with respective glycoconjugate ligands, [54][55][56] integrins with respective ligands, [35,[57][58][59] T cell receptor and coreceptor (TCR) with peptide-major histocompatibility complexes (pMHC), [19,[60][61][62][63] and Fc gamma (Fcγ) receptors with immunoglobulin G (IgG) Fc. [53,54,[64][65][66][67] In the micropipette aspiration assay, the ligands of interest are coated onto an RBC. Controlling for area and time, this coated RBC is directly brought into contact with another cell that expresses the opposing receptors to enable bond formation. [68] To achieve higher spatial and temporal resolution, a glass bead was attached to the RBC apex to modify this assay in the context of BFP ( Figure 2). While the micropipette assay only visually identifies binding events, BFP offers the advantage of real-time tracking using a high-speed camera. Thus, BFP is preferred for adhesion frequency measurement in cases where the off-rate is greater than 5 s −1 . For instance, Huang et al. employed BFP to perform an adhesion frequency assay, in which a T cell was brought into contact with the opposing bead, controlling for time and contact area. [19] As the interaction between TCR and pMHC is crucial for the immune response, kinetic analysis of this interaction is of interest. [69] In this adhesion assay, the probability of adhesion was assessed based on the frequency of adhesions (P a ) observed in 50 repeated contact cycles using a single pair of beads/cells ( Table 2).
where P a is the adhesion frequency, and where m r and m 1 are receptor (e.g., TCR) and ligand (e.g., pMHC) densities, A c and t c are contact area and time, and K a and k off are 2D binding affinity and off-rate. The 2D onrate can be calculated from k on = K a × k off . Since its advent, this BFP adhesion assay has been applied to other cell types, including platelets [29,70] and neutrophils. [15,71]

. Thermal fluctuation assay
In addition to the adhesion frequency assay, the thermal fluctuation assay is another method used to measure 2D binding kinetics of ligand-receptor pairs ( Figure 2). While the adhesion frequency assay extracts 2D kinetic parameters from a probabilistic model and reports binding frequency as a function of contact time, the thermal fluctuation assay employs a force sensor to detect bond formation and dissociation by monitoring changes in thermal fluctuations. [72] This assay is based on the premise that force probes like BFP are typically susceptible to thermal fluctuations. [72] In fact, the method initially proposed by Chen and Evans to monitor 2D ligand-receptor interactions was based merely on thermal fluctuations of the BFP probe. [55] They utilized the BFP to monitor the interactions between P-selectin glycoprotein ligand 1 (PSGL-1) coated on the probe bead and L-selectin or P-selectin on the target bead.
TA B L E  Essential mechanical variables in BFP dynamic force spectroscopies. This table presents a range of customizable parameters during BFP experiments. Columns show parameters, parameter definitions, force versus time curve representation, and schematic representation of key variables. Row 1 presents the contact time (t c ) of 0.1 s (magenta) and 0.2 s (green). Row 2 presents the impingement force (f c ) of 20 pN (magenta) and 50 pN (green) and Row 3 presents the ramping rate (r f ) of 10 2 pN s −1 (grey), 10 3 pN s −1 (magenta), and 10 4 pN s −1 (green).

Contact time t c [s]
The duration for the target bead/cell to stay in contact with the probe bead.
The magnitude of force that the target bead/cell impinges the probe bead at.
Ramping rate How fast the target bead/cell retracts after impingement.

. Force-clamp DFS assay
BFP can also be used under a range of constant forces to measure single ligand-receptor bond lifetimes. This assay is termed a force-clamp assay and has been utilized by several groups studying both platelets [25][26][27]50] and T cells. [33,51] In a recent study, Chen et al. utilized the force-clamp assay to elucidate distinct state transitions of platelet integrin α IIb β 3 during platelet aggregation. [50] Platelets play a critical role in hemostasis and thrombosis, which are heavily mediated by integrin α IIb β 3 . [73] In this assay, a platelet was manipulated to contact and impinge on a probe bead coated with fibronectin (FN), then retracted at a constant rate (3 μm s −1 ). If binding was detected during retraction, the target pipette was held at a set force to await bond dissociation. Using platelets again, Ju et al. employed the BFP forceclamp assay and found that in glycoprotein Ibα (GPIbα), multiple leucine-rich repeats (LRR) can be unfolded under force. This unfolding starts from the noncontact LRR2-4, and the observed force-strengthened bond behavior, where forces in the range of 10-25 pN strengthened individual bonds between von Willebrand factor A1 domain (VWF-A1) and platelet receptor GPIbα, is consistent with their previous reports. [26,29,74] This suggests that the unfolding events allow for a better fit of the A1 domain into the enlarged GPIbα binding pocket and thus prolongs the lifetime of the bond. [26] Further, this provides a potential explanation for platelet agglutination via VWF-GPIbα alone under pathological shear (>10,000 s −1 ). [75] Furthermore, the GPIbα juxta membrane mechanosensitive domain (MSD) was demonstrated to unfold under force and trigger α-type intracellular Ca 2+ signaling, while LRRD unfolding intensified Ca 2+ signals. Intriguingly, a cytoplasmic adaptor protein 14-3-3ζ was found to function as a signal transducer, transmitting force on the VWF-GPIbα bond to the MSD, providing a coupling between the two unfolded domains [25] (Table 4).

. Force-ramp DFS assay
Another key assay performed using BFP is the force-ramp assay ( Figure 2). In the force-ramp assay, the adhesion frequency assay is run for a pre-set contact time, and the occurrence of adhesions is determined at the end of this TA B L E  Key BFP measurements. The table lists measurements (Column 1), force versus time curve representations (Column 2), schematic representations (Column 3), and data analysis details (Column 4). A touch cycle shows the target cell/bead impinging the probe bead on the RBC, causing RBC deflection in the x-plane. The impingement is represented by a negative force (Column 2, magenta). When the target cell/bead is retracted, the RBC is pulled if a bond has formed between the ligand-receptor pair. This manifests as a positive deflection in the x-plane and is represented by a position force (Column 2, blue). In an adhesion event, the bond between the ligand-receptor pair quickly dissociates, and the force returns to its starting position (x = 0) and returns to baseline (F = 0 pN) quickly (Row 1). If a membrane tether forms, the cell membrane is separated from the cytoskeleton as the crossover force f ⊗ is reached (Row 2). The time duration in which the tether is maintained is the bond lifetime (Row 3). time. [50] For instance, Liu et al. used the force-ramp BFP assay with T cells, pulling a T cell at a constant speed until bond rupture. [33] Evans et al. employed two modes of force spectroscopy with the BFP force-ramp assay: a conventional "steady ramp" and a novel "jump ramp." They discovered that two pathways with significantly different kinetics exist. Starting from zero force, they applied slow and steady ramping rates to the cell. In doing so, they observed that P-selectin-PSGL-1 bonds were weak and readily broke upon small force application. This pathway was characterized as a "low impedance failure pathway with a fast dissociation rate." Conversely, when cells were pulled in the same manner under fast ramping rates, P-selectin-PSGL-1 bonds strengthened, with breakage occurring at increasing forces. This pathway was identified as a "high impedance failure pathway." Labelled as a "catch-slip" bond, they found that small force could initially lead to bond strengthening, and subsequently exposing that bond to increasing forces could then accelerate bond failure as the principal energy barrier is lowered [76] (Table 4).

. Multimode assays
The BFP is a highly versatile tool that can be utilized in an array of contexts. Table 3 showcases key events that can be monitored with a combination of the BFP assays described in this review. By integrating several of the assays described earlier, Ju et al. observed "biphasic force decelerated (catch) and force accelerated (slip) dissociation" of GPIbα from VWF on platelets [29] (Table 4), confirming previous experiments demonstrating this catch-slip phenomenon. [74] Used as an in-house setup with LabVIEW programs for image analysis in combination with a piezoelectric translator control, they measured adhesion frequency and bond lifetime through repeat impingement cycle experiments. At nonzero forces, a force-clamp assay was used, while at zero force, the thermal fluctuation assay was preferred. [29] Moreover, Chen et al. employed a combination of forceclamp and thermal fluctuation experiments to demonstrate intercellular adhesion molecule 1 (ICAM-1) binding to lymphocyte function-associated antigen 1 (LFA-1) in different conformations, including the bent conformation with the lowest affinity state. Through these experimental assays, they revealed how force influences LFA-1 conformations and subsequently regulates its kinetics with ICAM-1 (Table 4). Lifetime distributions identified three different states with distinct off-rates. Application of force produced catch bonds at the lower forces, as fractions were shifted from the short to intermediate and long-lived states. However, at high forces, off-rates increased exponentially, as catch bonds are converted TA B L E  Ligand-Receptor binding events and representative BFP force versus time curves. The table shows different types of ligand-receptor interactions that can be characterized using BFP, along with schematic representations and illustrative force versus time curves. Column 1 lists the type of interaction, while column 2 shows the force versus time curves, and column 3 provides the corresponding schematic. The interactions include ligand-receptor binding, receptor unfolding, cooperative binding, bending, and unbending.
• Observed the unfolding of the LRRD and MSD of GPIbα • Discovered that VWF activation can be induced via hemodynamic force • Magnitude and duration of force are important to prolong pMHC-TCR bond lifetime and lead to conformational change • Revealed the dynamic mechano-chemical coupling mechanism of pMHC-TCR catch bond.
• Shed light on T cell selection in adaptive immunity mechano-regulation • Observed a three-step of P-selectin-PSGL-1 bond dissociation • Observed real-time reversible conformational switch of LFA-1 between bent and extended. LFA-1 conformation and force regulate the ICAM-1-LFA-1 kinetics.
• Discovered enhanced LFA-1-ICAM-1 catch bond behavior at 10 pN to slip bonds. [77] Similarly, thermal fluctuation and forceclamp experiments were used to quantify how dissociation of LFA-1 from ICAM-1 is mediated by the initial and subsequent conformations of LFA-1, providing new insights into how cell adhesion and signaling is mediated by integrin function. [78] Lastly, T cell signaling, and adaptive immunity is regulated by the ability of TCR on T cells to recognize pMHC complexes on antigen presenting cells. Wu et al. used the thermal fluctuation and adhesion frequency assays to test whether pMHC-TCR catch bonds were affected by force-enhanced or force-induced H-bonds at the pMHC-TCR binding interface ( Table 4). The relationship between human leukocyte antigens (HLAs) and cancer has been intensively investigated, and cancer-associated somatic mutations in HLA-A2 have been identified previously. They demonstrated that agonist pMHC conformations were induced by mechanical force. Significantly, this was crucial for activation of both mouse and human pMHC-TCR catch bonds, which leads to both amplification of TCR antigen discrimination and T cell function initiation. Finally, they found that whenever cancer-associated somatic mutations restricted pMHC conformational changes, pMHC-TCR catch bonds were suppressed. [79]  BFP UPGRADES

. Dual BFP (dBFP)
Next, we would like to detail two key BFP upgrades, the first of which is the development of a dual BFP system (dBFP), represented in Table 5. Ju et al. developed the dBFP system, which uses two probe-and two target-micropipettes and enables the analysis of dual receptor crosstalk on a single cell in a step-by-step manner. [82] The signal initiated upon one receptor binding will travel over a distance to activate the other receptor. Temporal crosstalk involves presenting two ligands by two separate probes at distinct time points, allowing the signal initiated upon the first ligand-receptor binding event to upregulate another receptor. The power and TA B L E  Dual BFP configurations and applications. Column 1 lists the application, while column 2 provides the corresponding schematic, and column 3 lists the key findings of the application. The applications include using two probe beads to interrogate two receptor species' temporal crosstalk on a platelet [27,50,85] (Reproduced with permission. [27] Copyright 2017, Springer Nature), and spatial crosstalk on a T-cell [86] (Reproduced with permission. [27] Copyright 2017, Springer Nature).

Application Thrombosis Immunology
Cell type Platelet T cell • The 'Switch' assay setting was then combined with compression assay to reveal the compression force regulation of on diabetic platelet

• Extended previous concept on pMHC-TCR interaction
• Revealed that the TCR-induced LFA-1 activation is a global process, where pMHC-TCR interaction triggers global and sustained upregulation of LFA-1 binding affinity utility of this dBFP was nicely demonstrated in four important dual receptor systems: (TCR/LFA-1), (GPIbα/α IIb β 3 ), (GPIbα/CD62p), and (P 2 Y 1 /CD62p). [27] The dBFP is like the conventional BFP, configured with four micropipettes instead of three. For example, Pang et al. utilized a modified dBFP method to primarily stimulate integrin α IIb β 3 and subsequently report phosphatidylserine (PS) exposure. [82] In this study, the stimulating BFP probe was coated with FN and the test micropipette with the platelet was first brought into repeated contact and retraction with the FN beads for a period of 5 min. Afterwards, the platelet was quicky realigned to the reporting micropipette for measurement of annexin V binding to report PS exposure. This new technology allowed Pang et al. to correlate platelet activation, specifically activation of integrin α IIb β 3 , with PS exposure. Chen et al. also utilized the new dBFP technology with platelets. They utilized a VWF-A1 probe to stimulate a platelet that was then switched to a FN or fibrinogen (FBG) coated probe. As such, they measured integrin α IIb β 3 adhesion frequency after the first VWF-A1-GPIbα lifetime event regardless of its duration. [50] As a result, they found that a mechanical dependency of integrin α IIb β 3 activation exists and that the post switch adhesion frequency of integrin α IIb β 3 increased with the A1 lifetime. These findings support the role of integrin α IIb β 3 in arresting platelets from translocation under high shear [83,84] (Table 5). Ultimately, this novel upgrade in BFP enables the quantification of the "spatiotemporal requirements" and reveals the "functional consequences of the up-and down-stream" signaling events. [8]

. Fluorescence BFP (fBFP)
Another influential BFP technical advancement was the development of fluorescence BFP (fBFP) described in detail by Chen et al. [70] and is represented in Table 6. When combined with fluorescence imaging, BFP-DFS correlates the force experienced on a cell with ligand-receptor binding kinetics and subsequent intracellular calcium signaling. For example, Chen et al. used the fBFP system in combination TA B L E  Fluorescence BFP configurations and applications. Column 1 lists the application, while column 2 provides the corresponding schematic, and column 3 lists the key findings for each application. Some of the applications include using fBFP to observe the intraplatelet Ca 2+ flux during the GPIbα unfolding on a platelet, [26,27] and to observe intracellular Ca 2+ levels alteration in T cells upon repetitive TCR-pMHC binding on a T cell. [89][90][91]

Application Thrombosis Immunology
Cell type Platelet T cell Additionally, Ju et al. combined the fBFP with their novel dBFP setup to monitor Ca 2+ signaling in platelets upon GPIbα activation. [27] Remarkably, they were able to directly correlate Ca 2+ signaling with binding events and bond lifetimes on single platelets during GPIbα activation. Further, fBFP was also utilized to monitor adenosine diphosphate (ADP) binding to P 2 Y receptors on platelets and track platelet activation through Ca 2+ increases. [27] Similarly, Liu et al. utilized the force-clamp BFP assay with T cells in conjunction with fBFP. [33] In this study, T cells from OT1 transgenic mice were utilized. A range of constant forces were applied via a pMHC engaged to a TCR on the T cells and 2D single-bond lifetimes were measured by a force-clamp assay in repetitive cycles. Multiband formation was minimized by ensuring a brief contact time (0.1 s). Adhesion frequencies were kept low (<20%) in order to ensure that binding events were mediated by single bonds. To do so, the pMHC density on the probe bead was adjusted accordingly. Interestingly, fBFP was used simultaneously to measure bond lifetime and Ca 2+ flux. Further, force was correlated with maximal percent of Ca 2+ increase to assess how force regulation impacts T cell triggering. [33] In summary, coupling of fluorescence imaging with the conventional BFP setup has provided researchers with the ability to monitor membrane ligand-receptor interaction and cellular signaling simultaneously (Table 6).
Meanwhile, other than the fBFP, there are other existing fluorescence-labelled force sensor platforms used for cell detection. [87] Dutta et al. have introduced molecular-tensionbased fluorescence microscopy (MTFM) with the usage of DNA origami hairpin structure as force sensors that demonstrates a proportional correlation between applied mechanical forces and fluorescence intensity it emits. The changes of the detected fluorescence intensity can further reflect the direction and magnitude of the applied forces. [88] The DNA tensioner platform, another recent technique developed by TA B L E  Modified BFP configurations and applications. Column 1 lists the application, while column 2 provides the corresponding schematic, and column 3 lists the key findings for each application. Some of the applications include using a modified dual BFP setup to activate platelets with adenosine diphosphate (ADP) before engaging a soluble and an immobilized ligand on the probe [17] (Reproduced with permission. [27] Copyright 2017, Springer Nature), and using an ultra-stable BFP to achieve higher accuracy on force determination for bond lifetimes beyond 200 s in force clamp assay [46,90] (Reproduced with permission. [46] Copyright 2020, American Chemical Society).

Upgrades
Replace probe with an agonist reservoir Ultra-stable BFP

Application Thrombosis Cancer biology
Cell types Platelet T cell Ligand-receptor axes P 2 Y receptor, CD62p PD-1

BFP configurations
Significance • ADP stimulation of the P 2 Y agonist receptor upregulated the platelet activation marker CD62p and displayed higher binding to the anti-CD62p compared to the pre-stimulated platelets • Provided evidence for signaling events can be initiated by soluble agonist binding to a surface receptor, hence upregulating the expression or function of another receptor • Developed and incorporated a smart control feedback system into the force clamp assay to measure bond lifetimes beyond 200s, resolved the probe drifting issue in the conventional setup, leading to more accurate force determination • Combined with MD simulation to predict the protein-protein interaction binding site, leading towards the new era of single cell mechanobiology study adapting the concept of TFM and further combined with the implementation of microfluidics design, enabling the detection of mechanical force distribution while allowing for high throughput operation. [87]  BFP APPLICATIONS

. Immunology
BFP has significantly contributed to the field of immunology and cancer biology (Table 7) by providing biomechanical insights. Recently, An et al. utilized BFP to benchmark the dissociation kinetics of three clinically approved monoclonal antibodies (mAbs) that target programmed cell death protein (PD-1) on T lymphocytes. An ultra-stable BFP force-clamp assay was developed. As such, they were able to measure long bond lifetimes (>200 s) while ensuring stable and accurate clamped holding forces. In doing so, they were able to precisely characterize mAb-immunotherapeutic target binding kinetics at the single molecule level, suggesting a "kinetic platform to direct the screening, optimization, and clinical selection of therapeutic antibodies in the future." [46] Additionally, researchers have utilized fBFP technology to investigate the forces exerted on T cells during antigen recognition and activation, which is essential for adaptive immunity development in response to pathogens or tumor cells. [51] By combining BFP with fluorescent imaging, researchers have been able to simultaneously image cell morphology and Ca 2+ signaling. In a similar study, Liu et al. used fBFP to observe Ca 2+ signals induced by force in live T cells. [52] Moreover, Sawicka et al., employing a modified BFP method, revealed changes in Young's modulus of T cells during activation, demonstrating a cellular stiffening effect within the first minutes of the activation process. [92]

. Thrombosis
BFP has also been influential in the field of thrombosis, characterizing the kinetics of platelet receptors involved in the exaggerated responses of platelet adhesion and aggregation (Table 7). One key platelet receptor studied quite extensively using BFP is GPIbα. Ju et al. showed that GPIbα conformational changes enhance binding to VWF-A1. [26] Similarly, BFP was used extensively to demonstrate the role of force transduction on platelet surface reactivity. [25] Additionally, BFP was used to characterize the force-dependent kinetics of GPIbα dissociation from the VWF-A1 domain of different N-terminal lengths immobilized on different surfaces. Ultimately, these findings helped to explain the "four phases of collagen dependent enhancement of VWF-GPIbα interaction" under flow, providing novel insight into platelet dynamics during thrombotic response. [30] Moreover, BFP has been used to explain the mechanobiology of another key platelet receptor: integrin α IIb β 3 . Interestingly, Xu et al. employed BFP to detect direct interactions between apolipoprotein A-IV (apoA-IV), which is an abundant plasma lipid binding protein inversely correlated with cardiovascular disease, and integrin α IIb β 3 . Through their work, they identified apoA-IV as an endogenous inhibitor of thrombosis. [28] More recently, studies have demonstrated the existence of a distinct prothrombotic phenotype in diabetes which is regulated by a compression force sensing mechanism linked to α IIb β 3 adhesive function. Historically, it has been established that diabetic patient platelets are more reactive than nondiabetic platelets. BFP studies have attributed this increased reactivity in part to α IIb β 3 dysregulated compression force sensing in diabetic patients. [85] BFP technology, combined with other methodologies, has played a crucial role in identifying the integrin α IIb β 3 intermediate state's role in promoting biomechanical platelet aggregation. [50] . Inflammation BFP has significantly contributed to our understanding of the inflammatory response. [93] Evans et al. have worked at length on utilizing BFP to understand neutrophil mechanobiology at the ligand-receptor scale. A series of articles published by Evans' group characterized and quantified the bond kinetics of PSGL-1 and P-selectin, which are essential for neutrophil recruitment to the endothelium and play a fundamental role in the immune response. [20,21,37] Briefly, this series of studies probed neutrophils with P-selectin coated beads and quantified the forces experienced by interaction during retraction of the neutrophils, providing valuable insight into neutrophil recruitment dynamics. Additionally, BFP has been used to investigate another key neutrophil receptor: LFA-1. Integrins are thought to play a major role in mechanosensing and mechanotransduction by transmitting forces and transducing signals across the cell membrane. BFP has revealed catch bond kinetics for ICAM-1-LFA-1 binding, [49] and the influence of both outside-in and inside-out signaling on ICAM-1-LFA-1 bond lifetimes. [15,16] Finally, in combination with a flow chamber, BFP was used to explain a triphasic force dependent of E-selectin-PSGL-1 dissociation on rolling neutrophils. [14] Overall, these molecular insights have helped elucidate neutrophil tethering, rolling, and subsequent adhesion on vascular surfaces, which has widespread implications in the context of inflammation.

 CONCLUSION AND FUTURE PERSPECTIVES
BFP has emerged as an incredibly powerful technique for investigating cell mechanobiology in vitro. Over the past few decades, this technology has evolved substantially, progress-ing from a force probe and imaging modality to encompass fluorescence imaging and dual receptor crosstalk investigation. In comparison to other DFS nanotools available, BFP offers benefits in force resolution and its compatibility with live cells. While positional resolution with BFP is 10× lower than that which can be achieved with AFM, force resolution is substantially higher with a detection of ≈3 pN at ≈30 nm spatial resolution. Unlike AFM, which is typically performed on cells adherent to a coverslip or cantilever surface, BFP uses a micropipette in a buffer filled chamber which holds cells gently in a nearly physiological environment. [94] Further, in contrast to techniques such as micropipette aspiration and AFM indentation which induce deformations by applying surface forces to the cell in a non-discriminatory manner, using ligand coated micrometer-sized beads allows the mechanical loads to be selectively applied to intracellular structures via specific receptors. [95] Another mechanical force probing technique, which can be applied to a broad range of mechanical systems is traction force microscopy (TFM). [96] Unlike BFP, TFM has no inherent size or force scale and has significantly higher throughput. However, while TFM is versatile and easy to use, it is typically utilized at measure forces over macro-scales. BFP on the other hand, offers more precise measurements over nano-and micro-scales. [97] Comparisons of the various DFS techniques have been reviewed and summarized previously and can be easily referred to. [7,98]

. Limitations
Nevertheless, challenges and limitations still exist in the applications of the BFP. While the BFP offers high sub-piconewton force resolution with an adjustable loading rate, a nanometerscale spatial resolution, and good temporal control with milliseconds of resolution, it has relatively low throughput. [99] Additionally, it is time-and cost-intensive as only one pair of ligand-receptor interactions can be characterized at a given time. The measurements with the BFP are limited to pulling forces and displacements smaller than ≈0.3 μm [92] and the cell of interest aspirated by the micropipette may experience a change in shape over time. [99] .

Developments in DFS
The continued development and implementation of mechanical molecular nanotools combined with optical techniques have steadily advanced knowledge in single cell biomechanics. Nevertheless, there is a variation in these nanotools' throughput, sensitivities, and spatiotemporal resolutions, as well as an emerging need for characterizing and visualizing cell mechanosensing. [98] Additional advances in DFS have begun to integrate force spectroscopies with microfluidic systems to create a lab-on-a-chip platform to simulate the multicellular microenvironment of human-specific physiology and pathophysiology. For example, the OT-integrated microfluidic system has demonstrated its ability in sorting and/or manipulating biological molecules. [100][101][102] More importantly, a microfluidic-integrated micro clot-array-elastometry system has recapitulated the dynamics of platelet clot biomechanics under hemodynamic shear force and biochemical treatments [103] highlighting its potential in identifying therapeutic targets, translating into a point-of-care system for coagulation diagnosis and a high-throughput antiplatelet drug testing platform. On the other hand, to improve the throughput of micropipette-based techniques such as BFP, an automated operational procedure for using parallel arrays of serial micropipettes on a microfluidic platform has been established. [104,105] This parallel setup has demonstrated the ability to capture multiple dynamic measurements of individual cells simultaneously, where each cell is sampled multiple times by the serial micropipettes to assess consequential effects. Therefore, parallelization enables the user to appreciate cell dynamics and variability and offers higher throughput for laboratory research and potential clinical drug testing and screening. Force spectroscopy traces are often acquired at a high acquisition rate to capture dwells in transient dynamics and short-lived molecular states. However, slow response of the single-molecule force spectroscopy can distort the signal and lead to misinterpretation of results by data-processing algorithms. [106] To address these response dynamics, an adaptation of Bayesian nonparametric computational algorithm offers high potential. It allows for clear physical interpretation and provides posterior probabilities for modelling complex biomolecules. [107] The algorithm was established to allow data acquired within the low kHz timeframe to be analyzed, and presents a clear interpretation of molecular dynamic transitions, instrumentation response, and noise. [106] Further, this adapted algorithm sees a potential for unsupervised time series analysis of cell adhesion and spreading when combined with single-molecule force microscopies [106] and optical microscopy such as super-resolution microscopy. [108]

 CONCLUSION
BFP is a precise nanotool for studying molecular interactions and cell mechanosensing, with potential applications in cancer, thrombosis, inflammation, and drug targeting. However, its accessibility remains a challenge. By developing user-friendly data processing interfaces and automating the tool, BFP's feasibility can be improved. Stable feedback control algorithms can further enhance BFP's force resolution, expanding its potential applications. Recent developments, such as the "BFPTool," have integrated and simplified BFP experiment image processing and analysis, making it more accessible to researchers. [109] BFP has also shown potential in clinical applications, elucidating monoclonal antibodies' dissociation kinetics, [46] platelet receptor kinetics for potential drug targeting, [26,27,30,85] and neutrophil recruitment in inflammation. [21,37] With the growing interest in the field of cell mechanobiology, BFP is expected to continue expanding as a cutting-edge technology for studying biology at the single-cell level. By improving userfriendliness and clinical relevance, BFP technology can advance our understanding of cellular mechanics and contribute to novel therapeutic discoveries. The development of stable feedback control algorithms, [46] automation, and userfriendly data processing interfaces can increase its output and efficiency. [51,110]