(a,b,c) Violin plot shows individual phagocytic events (colored markers, n = 23), mean (black cross), and median (dashed line). (a) Engulfment time for individual phagocytic events. (b) Maximum target constriction by live-cell imaging using lattice light-sheet microscopy (LLSM). For each movie, the single time point in which target constriction was maximal was identified, and the exact constriction value was determined as described in Figure 3—figure supplement 1. (c) Average target constriction at 50% engulfment. Average is 0.25 ± 0.04 μm (s.e.m., n = 23 cups), which is ~40% larger than observed with fixed cell data at similar stage at 0.18 ± 0.02 μm (s.e.m., n = 19) (Figure 2i). This indicates that partial relaxation of the particle to a more spherical shape occurs during fixation, and that the fixed cell force measurements are likely a slight underestimate of the real phagocytic forces. (d) Volume of deformable acrylamide-co-acrylic acid-microparticles (DAAMPs) decreases with phagocytic internalization. Since hydrogel microparticles are not completely incompressible, their volume can decrease under exertion of bulk forces (Vorselen et al., 2020b). Same event is shown as in Figure 1a and d. Colorscale denotes radial distance to the centroid. Time stamps are provided in min:s, and internalization is complete at the 3:58 time point. Scale bar, 3 μm. (e) Quantification of effective particle diameter, bulk stress, and sphericity over time for DAAMP in (d) Bulk compressive forces can be estimated from the previous DAAMP bulk modulus measurements (~3.8 kPa) (Vorselen et al., 2020b). Gray area indicates time interval of phagocytosis. Compressive stresses arise during phagocytosis and are increased after completion of internalization. (f) Quantification of bulk stresses, target sphericity, and displacement of 23 live-cell phagocytic events. Compressive stresses are exerted during phagocytosis (~0.5 kPa) and increase after completion (~1.3 kPa). Particle sphericity dips during phagocytosis, but the particles return to a more spherical shape after internalization completion. Gray area indicates the duration of phagocytosis, where normalized time t = 0 indicates the start of the phagocytic event, and t = 1 internalization completion for individual events. Particle diameter and sphericity (because it could be strongly affected by imaging artifacts, see h) were normalized to 1, using the measurements before the start of phagocytosis. (g) Brief F-actin accumulation (orange arrow) observed on DAAMP phagosome following internalization. Time lapse montage (min:s) of RAW macrophage transfected with mEmerald-Lifeact internalizing a DAAMP (9 μm, 6.5 kPa) functionalized with IgG and AF647-Cadaverine and imaged using LLSM. Scale bar, 5 μm. (h) Typical LLSM images showing artifacts that hinder particle reconstruction and force analysis. Artifacts are not obvious in the xy-plane, but a strong ‘striping’ artifact, with strong fluctuations in fluorescent intensity (yellow arrows) are visible along the optical axis. Shape reconstruction in microparticle traction force microscopy (MP-TFM) is critically dependent on particle edge detection, which shows clear irregularities (yellow arrows) because of the striping artifact. Such artifacts result in large apparent particle deformations inside and outside the cell-target contact area, and can make it impossible to converge on a solution when solving the elasticity theory problem to infer tractions from target deformations. Because of this, we only used the confocal image data for force analysis. Scale bar, 5 μm.