10 mL of S. acidocaldarius cells from an exponentially growing culture were collected by centrifugation at 5000 g at room temperature. The pellet was washed once in phosphate buffered saline (PBS), resuspended in 225 μL PBS, and 25 μL of Alexa Fluor 568-NHS-ester dye (Thermo Scientific) freshly reconstituted at 0.5 μg.mL−1 in anhydrous DMSO was added. Cells were incubated with the dye for 15 min protected from light, with gentle shaking, at room temperature. Cells were then pelleted, washed twice with minimal Brock’s media, and recovered in 5 mL complete Brock’s media at 75°C with shaking for 1.5 h prior to imaging.
Cells were imaged in a VAHeat chamber controlled by the VAHeat system (Interherence) on a Nikon Ti-E inverted microscope equipped with a 100× 1.45NA PlanApo objective and an additional 1.5X intermediate tube lens, 561 nm laser, Hamamatsu Quest camera, and a VT-iSIM super-resolution module. Movies were recorded at a rate of two frames per second.
Cells were imaged in a VAHeat chamber controlled by the VAHeat system (Interherence) on a Nikon Ti-E inverted microscope equipped with a 100× 1.45NA PlanApo objective and an additional 1.5X intermediate tube lens, 561 nm laser, Hamamatsu Quest camera, and a VT-iSIM super-resolution module. Movies were recorded at a rate of two frames per second.