TABLE 1.

Summary of cellular force measurement techniques.

	Resolution (x/y/z/F/t)	Cell substrate	System requirements	Post-processing	Advantages	Disadvantages	References
Traction force microscopy (TFM) Figures 2A–C	Depends mostly on substrate properties and microscopy setup	Hydrogel, PDMS, elastomers, fibrillar matrices (e.g., collagen); Arrays of elastic micropillars	Standard fluorescent microscope; Confocal microscopy for out of plane bead tracking and 3-D tractions	Single particle tracking, correlation tracking, and/or particle image velocimetry; Theoretical/computational solid mechanics analysis	Cell substrate can be physiologically realistic (except micropillar arrays); Image based – highly versatile; Simple experimental setup and high throughput; Can be extended to provide collective cellular force measurements (e.g., monolayer stress microscopy)	Requires zero force state (except micropillar arrays) and calibrating substrate elastic properties; Limited sensitivity to vertical forces; Fluorescence microscopy over long periods can cause phototoxic effects	Tan et al., 2003; Wang and Lin, 2007; Fu et al., 2010; del Alamo et al., 2013; Hall et al., 2013; Style et al., 2014; Polacheck and Chen, 2016; Serrano et al., 2019
Atomic force microscopy (AFM) Figure 2D	Resolution depends on the imaging force and probe geometries; Lateral resolution 1–1.5 nm; Vertical resolution 0.1 nm; Force resolution 100 pN	Mica, glass, or glass slides modified with Silane to enhance cell adhesions	Piezoelectric scanner for mounting samples; Proper probes attached to pliable silicon or silicon nitride cantilever; Laser beam/photodiode setup for measuring cantilever deflection	Cantilever deflection as a function of vertical displacements; Conversion a force-versus-separation distance curve	Probes for molecular interactions, physiochemical properties, surface stiffnesses, and macromolecular elasticities	Requires careful sample preparation and data collection; Requires physical contact between the AFM probe and the sample – cannot probe basal structures (e.g., podosomes tips) Localizing specific cell structures (e.g., podosomes) by AFM alone is challenging	Labernadie et al., 2010
Protrusion force microscopy (PFM) Figure 2E	The same as AFM; Vertical resolution 10 nm; Line rate on order of 1 Hz; Force resolution to the order of nN	Compliant formvar membranes	AFM system and fluorescence microscopy	The same as AFM, plus mathematical model to infer podosomes forces from formvar membrane deformation	Measures protrusive forces applied perpendicularly to the substrate at a single podosome level; High spatiotemporal resolution	The same as AFM, except for localizing podosomes; Narrow range of applications.	Labernadie et al., 2014; Bouissou et al., 2017
Elastic resonator interference stress microscopy (ERISM)Figure 2F	Displacement resolution 2nm (limited by surface); Temporal resolution <0.5 s; Lateral resolution ∼1.6 μm;	Elastic optical micro-cavity comprized of a layer of ultra-soft siloxane-based elastomer sandwiched between semi-transparent gold layers	Conventional wide-field phase contrast or fluorescent microscopy with a tunable light source capable of providing monochromatic illumination	Each light fringe ∼ 200 nm = ≥ count fringes to determine size of deformations; Conversion of forces by utilizing substrate mechanical properties	Unlike many TFM methods, no zero-force state required; No phototoxic effects; Versatile, and compatible with other microscopy methods; Excellent vertical and lateral force sensitivities	Experimental setup and fabrication of ERISM cavities are relatively involved; 2D soft substrate may not be physiologically realistic for some applications	Kronenberg et al., 2017; Liehm et al., 2018
Molecular tension-fluorescence lifetime imaging microscopy (MT-FLIM) Figure 2G	Force threshold F1/2 is a measure of the applied force at which 50% of probes are open, estimating applied force ranges; Force resolution to the order of pN exerted by individual integrins	Supported lipid bilayer (SLB) – phospholipid membranes; Confined in the Z-direction but are laterally fluid	Inverted microscopes with perfect focus capabilities and appropriate lasers for excitation; Specific software required;	Matlab Bioformats Toolbox and semiautomated custom scripts; FIJI plugins, including MultiKymograph and TrackMate	Highly specific observations of integrin behavior and force generation as it relates to podosome formation and mechanosensing	Highly technical in the development and implementation of molecular tension probes, and in microscopy set-up Misses forces transmitted via non-specific interactions	Wang and Ha, 2013; Blakely et al., 2014; Zhang et al., 2014; Brockman et al., 2018; Glazier et al., 2019
Microsphere-based traction force microscopy Figure 2H	Force resolution to the order of nN	Hydrogel-based microspheres; Fabricated by water-oil emulsions	Standard fluorescent microscope; Confocal microscopy for tracking 3-D shape deformations	Comparison between deformed and undeformed states from 3-D shape reconstructions	Suitable for studying forces in environments with complex mechanical properties, where TFM and ESRIM would be challenging	Intensive image processing requirements Resolution is limited by spatial distribution of microspheres in sample	Girardo et al., 2018; Mohagheghian et al., 2018; Kaytanli et al., 2020; Vorselen et al., 2020b