Table 2.

Trouble-Shooting Traction Force Microscopy

Concern	Mitigation Strategies
Poor cell attachment, cell death	Improve protein attachment: higher concentrations of functionalization chemicals and/or proteins; increased wash steps and/or wash times (SWA: §2.2, qTFM: §3.2.3) Improve environmental control: increase temperature and/or time for protein and cell attachment; reduce exposure time/intensity of fluorescence exposure; adjust media formulation (Supplemental Files §B.2)
Suspicious wrinkle counts (SWA)	Improve signal-to-noise ratio: Reduce hydrogel stiffness (§2.2); crop images and/or use regions-of-interest to exclude errant regions Leverage computational tools: Consider different analytical approaches (Supplemental Files §A); exclude wrinkles likely to represent noise (e.g. less than 4 px2 in area) and/or overexposed regions (e.g. greater than reasonable size determined by observation)
Randomly oriented displacement vectors (qTFM)	Improve signal-to-noise ratio: optimize fabrication of gels to ensure level and well-distributed field of markers (Supplemental Files §B.1); enhance image focus (Supplemental Files §B.3); reduce hydrogel stiffness (§3.2.1); reduce size of IR (§3.4.2) Leverage computational tools: crop images and/or use regions-of-interest to exclude errant regions; pre-process images (Supplemental Files §B.4); pre-align images before calculating displacements (§3.3.2); apply vector validation or ensemble average to smooth vectors (§3.4.3)
Suspicious force values (qTFM)	Check units: ensure displacements, forces (e.g. N), and stresses (e.g. Pa = N/m2) are all converted correctly from pixels with appropriate orders of magnitude (§3.5) Improve cell health: see recommendations above Inspect displacement data: poorly adhered cells, stiff substrates, and large interrogation regions (IRs) could lead to displacement quantification near the noise floor and subsequently lower-than-expected forces; randomly assigned vectors can result from regions that have no beads or beads that move out of frame can result in higher-than-expected forces, so ensure beads are well distributed (§3.2.2), consider increasing size of IR (§4.3.2), and/or implement vector validation (§3.4.3) Reconsider microscopy parameters: increasing optical (not digital) magnification and/or improving resolution of acquired images will enable additional passes with smaller IRs and potentially better resolution of localized displacements and forces

SWA = Substrate Wrinkling Assay; qTFM = Quantitative Traction Force Microscopy