Live cell imaging of nuclear deformation and dynamics of Actin and Myo1f localization during migration in 3D environments. Analysis of 3D migration in a 1.5 mg/mL collagen gel toward an fMLP (100 nM) gradient was performed with dHL-60-EGFP-Myo1f cells or isolated human neutrophils. (A) Live cell imaging of the human neutrophil nucleus (labeled with Hoechst 5 µM, green) and the collagen meshwork (fire and red) using confocal reflection/fluorescence microscopy. Serial images of a movie of a representative neutrophil 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 neutrophil squeezes through a narrow pore. Arrows indicate the pore (white). Neutrophil localization is normalized to the position of the pore. Scale bar, 10 µm. The cell is representative for a total of 12 cells from 3 independent experiments. (B) Live cell imaging of a migrating dHL-60-EGFP-Myo1f cell using spinning-disk confocal microscopy. Pseudo-colored images demonstrate the morphology of the nucleus (gray) of dHL-60 EGFP-Myo1f cells, the subcellular localization of Actin (red) and Myo1f (green), as well as the colocalization of Actin and Myo1f (arrowheads). Arrows indicate constriction site. Scale bar, 10 µm. Color scales, heat map. Triangle indicates orientation of fMLP gradient. The cell is representative for a total of 9 cells from 3 independent experiments. (C) Schematic outline of the nuclear morphology (black), localization of Actin (red) and Myo1f (green), and colocalization of Actin and Myo1f (yellow) during 3D migration at indicated time points. The border of the cell body is depicted in gray.