Plan apo 100 1.49na tirf objective

Laser scanning confocal images were captured using Nikon Elements software on a Nikon A1R confocal microscope using a PlanApo λ100X 1.45 NA objective lens (Nikon). Parameters were set to satisfy the Nyquist criterion. Spinning disk confocal microscopy was performed on an Andor XDI Revolution (Andor Technology). The cells were imaged with a Nikon Plan Apo 100×1.49NA TIRF objective every 4 minutes. Images were acquired using MetaMorph with an Andor Neo cMOS camera.

For SIM imaging cells were imaged on a Nikon N-SIM system using a Nikon Plan Apo 100×1.49NA TIRF objective. Images were captured using Nikon Elements software with an Andor iXon3 camera. Z-step size was set to 0.100 um (well within Nyquist criterion). 15 images per slice (5 phases, 3 angles) were captured and then reconstructed using the Nikon Elements software. To minimize saturated pixels for quantification of the myosin spacing in different structures, we obtained images of the same field with various exposures. Images are displayed as extended depth focused maximum intensity projections. Sarcomeric distances were measured using intensity profiles drawn along the invasion pore. The intensity profiles passed through the center of each myosin head and spacing between peaks on the intensity profile indicated sarcomeric spacing. Statistical significance was determined by performing an ordinary one-way ANOVA in combination with Tukey's multiple comparisons in GraphPad Prism.