Table 1.

Summary of each TFM-based cellular force measurement analysis

TFM Methods	Target Forces	Dimension & Image acquisition	Substrate Materials	Advantages	Disadvantages	Refs
Deformable material-based 2D TFM	Cell-ECM	Cells on 2D substrates Force measurement in 2D Epifluorescence microscopy	PAA, PDMS, PEG	Simple experimental setups to prepare cell culture substrate Tunable substrate stiffness with wide ranges by concentration of monomers and cross-linking agents Scalable and economic Showing flat physiological surface Most popular and well verified method	Essential to have reference image without force for force analysis Required intensive image processing and stress computation steps Unable to measure normal (out-of-plane) forces	(27, 31, 32, 34)
Micropost-based 2D TFM	Cell-ECM	Essential to have reference image without force for force analysis Force measurement in 2D Epifluorescence microscopy	PDMS (microposts)	Tunable stiffness by geometrical parameters of microposts, such as diameters and heights Simple process for force analysis due to no need for reference image without force Higher degree of force sensitivity detected by bending of microposts	Required sophisticated photolithography techniques for substrate preparation Narrow range of stiffness Having discrete substrate morphology and less physiological surface due to the distribution of adhesion molecules Unable to measure normal (out-of-plane) forces	(36, 37)
Deformable material-based 3D (2.5D) TFM	Cell-ECM	Cells on 2D substrates Force measurement in 3D Confocal microscopy	PAA, PEG	Enable to measure normal (out-of-plane) forces, allowing to understand cell behaviors in 3D Simple experimental setups to prepare cell culture substrate Tunable substrate stiffness with wide ranges by concentration of monomers and cross-linking agents Flat physiological surface	Required highly intensive image processing and stress computation steps compared to 2D TFM methods Essential to have reference image without force for force analysis	(33, 41)
Deformable material-based 3D TFM	Cell-ECM	Cells embedded in 3D matrix Force measurement in 3D Confocal microscopy	PEG, type I collagen	Suitable to mimic in-vivo environment due to the 3D cell encapsulation Enable to measure normal (out-of-plane) forces, allowing to understand cell behaviors of 3D organoids in 3D Tunable substrate stiffness with wide ranges by concentration of monomers and cross-linking agents	Required the most intensive image processing and stress computation steps Essential to have reference image without force for force analysis, but it is difficult to acquire due to technical inability to remove cells within 3D substrate Complex force analyses due to the non-linear material properties (type I collagen)	(42, 43)