Myo1f^−/−nuclei failed to deform during 3D migration.Myo1f^+/+ and Myo1f^−/− neutrophils migrating in a 1.5 mg/mL collagen gel toward a CXCL1 (100 ng/mL) gradient. (A) Live cell imaging of the neutrophil nucleus (labeled with Hoechst 5 µM, green) and the collagen meshwork (fire and red) using confocal reflection/fluorescence microscopy. Serial images of movies of representative neutrophils migrating through a meshwork of collagen fibers at indicated time points. Confocal reflection images demonstrate the architecture of the collagen gel as well as the neutrophil body (fire and red). Fluorescence images depict the shape of the nucleus during migration within a collagen gel (green). Merge (yellow) shows the reflection image (red) and the nucleus (green). Schematic outline of the shape of the cell (red) and the nucleus (green) while the neutrophils squeeze through narrow pores. Arrows indicate the pores (white). Neutrophil localization is normalized to the position of the pore. Scale bar, 10 µm. The cells are representative for a total of 9 cells from 3 independent experiments. (B) Percentage of nuclear shape change during neutrophil 3D migration at indicated time points based on individual nuclear shape of each neutrophil during the observation period of 30 minutes using spinning-disk confocal microscopy. Time point 0 minutes represents the most spherical nuclear morphology of each individual cell that was observed for 10 minutes thereafter. n = 3 independent experiments; mean ± SEM; *P < .05; ***P < .001 (2-way ANOVA, Sidak multiple comparison test).