Plan 0.40 na air objective

High-resolution wide-field fluorescence images were acquired on a Nikon Eclipse Ti equipped with a Nikon 100× Plan Apo 1.45 numerical aperture (NA) oil ob­­­jective and a Nikon DS-Qi2 CMOS camera. Images were acquired every 10 s using 100- to 300-ms integration using a fluorescein isothiocyanate (FITC) filter cube. Photoconversion of NMIIA-mEOS2 was accomplished by closing the Eclipse Ti’s field diaphragm and using a 4’,6-diamidino-2-phenylindole filter cube to shine UV light on the cell’s leading edge for 10 s as previously described (Baker et al., 2010 (link)). This was immediately followed by opening the field diaphragm and imaging with FITC and Tex Red filter cubes every 1 min. Phase imaging was performed on a Nikon Eclipse Ti equipped with a Nikon 20× Plan 0.40 NA air objective and Nikon DS-Qi2 CMOS camera. Images were acquired every 1-min using 50-ms integration. A custom-built incubator was constructed to keep cells cultured in L-15 medium (BW12700Q; Fisher Scientific) at 37°C. An orange glass filter was placed on top of the condenser to prevent the inactivation of blebbistatin (Sakamoto et al., 2005 (link)).

Live-cell, wide-field phase microscopy videos were acquired on a Nikon Eclipse Ti, equipped with a Nikon 20× Plan 0.40 NA air objective and a DS-Qi2 complementary metal-oxide semiconductor camera. Images were acquired every 1 min. Cells in DMEM supplemented with 10 mM Hepes, were placed in a Tokai Hit stage-top incubation system during imaging at 37°C and 5% CO₂, and an orange, glass filter was placed on top of the condenser to restrict short wavelengths of light. Rates of ingression were calculated using ImageJ. Images were first aligned using the “StackReg” plugin, and a line of width 3 was drawn across the division plane of the dividing cells. Kymographs were generated using the “MultipleKymograph” plugin in ImageJ. Rates of ingression were then manually calculated from when the cell started to ingress until ingression had stopped, resulting in distance over time measurements.