Table 1. Overview of the simulation parameters used in this work.

Parameter	Value range
Substrate Thickness	100 μm
Substrate mechanical properties	E = 20 kPa, v = 0.49
Simulated Traction	5–50% E
FA diameter size	[0.5, 0.75, 1, 2, 3, 4, 5] μm
Bead density	[0.33, 1, 5, 10] Beads/(μm3)
Bead type	0.2 μm red-fluorescent beads (emission: 605 nm)
Bead location	3D (volumetric), 2D (surface)
Microscope simulation settings	Spinning-Disk Confocal with 60 × 1.4 Oil immersion lens. Pixel size (x, y, z) = [0.06 0.06 0.25] μm, ni = 1.51, ns = 1.4, NA = 1.4
Sample size	N = 20