1.2 na water immersion objective lens

Conventional widefield epifluorescence imaging was performed on an inverted Nikon Ti-S microscope configured with a 60 × 1.2 NA water-immersion objective lens (Nikon, Melville, NY, USA), a light emitting diode source (LED4D120, Thorlabs, Newton, NJ, USA), a multiband filter set (LF405/488/532/635-A-000, Semrock, Rochester, NY, USA) and images were captured with a Zyla 5.5 sCMOS camera (Andor, Windsor, CT, USA). The samples were illuminated 470 nm light at an intensity of ~2 W/cm² and with 200 ms exposures. For live cell experiments, samples were incubated at 37°C with Gibco CO₂ Independent Medium containing 50 µM DFHBI for 10 minutes prior to imaging. Time lapse movies were acquired over a period of 5 minutes with a 200 ms exposure every 5 seconds. For fixed cell imaging, samples were incubated at room temperature (~22°C) in PBS containing 50 µM DFHBI for 10 minutes prior to imaging.