1.49 na objective

Cells were imaged on either an Olympus IX-81 or Nikon Ti-2 inverted microscope fitted with TIRF optics. The IX-81 microscope used a 60 × 1.49 NA objective (Olympus) and an Orca Flash 4.0 sCMOS camera (Hamamatsu). Cells were illuminated with solid-state lasers (Melles Griot) with simultaneous acquisition by a DV-2 image splitter (MAG Biosystems). The microscope was maintained at 37 °C with a WeatherStation chamber and temperature controller (Precision Control) and images were acquired using Metamorph software. The Nikon Ti2 microscope was equipped with a motorized stage (Nikon), automated Z focus control, LU-N4 integrated four-wavelength solid state laser setup, TIRF illuminator (Nikon), quad wavelength filter cube, NI-DAQ triggering acquisition (National Instruments), an Orca Flash 4.0 sCMOS camera (Hamamatsu), and triggerable filter wheel (Finger Lakes Intstrumentation) with 525/50 and 600/50 wavelength emission filters. Cells were seeded on autoclaved 25 mm #1.5 round coverslips coated with 1 mL matrigel (80 µg/ mL) or recombinant Vitronectin-N diluted in PBS (Thermo Fisher). Cells were maintained at 37 °C with a stage top incubator (OKO Lab) and images were acquired with Nikon Elements.

TIRF microscopy was performed with the Ring-TIRF mode of the OMX V4 system described above, using a 60 × 1.49 NA objective (Olympus), using 488 nm and 561 nm laser lines. All other imaging conditions were as described above.