Here, 1 µl BODIPY (C9H7BN2F2) 493/503 from a 1 mg ml−1 stock in dimethylsulfoxide (stored in the dark) was added to 1 ml of culture in a 1.5-ml microfuge tube and incubated on a rocker (dark) for 30 min, centrifuged for 1 min at 2,000g and 950 µl of the supernatant removed. Cells were resuspended in the remaining media and 1.7 µl of the cell suspension applied to a slide for microscopy. Alternatively, cells from 3 ml of culture were centrifuged (20 s, 18,000g), resuspended in 50 µl of synthetic medium + 5 µl of 1:250 diluted autodot dye (Abcepta) and incubated as described above.
The microscope hardware, and image acquisition and projections from z-stacks were as reported previously47 (link), except the z-stack consisted of 25 images taken 0.35 μm apart, and Slidebook v.6.0.4 (Intelligent Imaging Innovations) was used. Alternatively, cells were imaged on a Nikon Eclipse Ti inverted microscope equipped with CSU-X1 spinning-disc confocal scan head (Yokogawa), 405-, 488- and 561-nm laser lines, 100× Apochromat total internal reflection fluorescence 1.4 numerical aperture objective (Nikon), Zyla 4.2 Plus sCMOS or iXon897 electron-multiplying charged-coupled device cameras (Andor) and NIS Elements AR software (Nikon).
The microscope hardware, and image acquisition and projections from z-stacks were as reported previously47 (link), except the z-stack consisted of 25 images taken 0.35 μm apart, and Slidebook v.6.0.4 (Intelligent Imaging Innovations) was used. Alternatively, cells were imaged on a Nikon Eclipse Ti inverted microscope equipped with CSU-X1 spinning-disc confocal scan head (Yokogawa), 405-, 488- and 561-nm laser lines, 100× Apochromat total internal reflection fluorescence 1.4 numerical aperture objective (Nikon), Zyla 4.2 Plus sCMOS or iXon897 electron-multiplying charged-coupled device cameras (Andor) and NIS Elements AR software (Nikon).