Ti inverted microscope

VDY6175, VDY6176, VDY6179, VDY6181, and VDY6186 cells were grown to saturation overnight in synthetic complete (SC) media containing 2% glucose. Cells were back-diluted to an OD600 of 0.1 in SC + 2% glucose and 1 μM beta-estradiol and grown for 3–4 hours to reach an OD600 ~0.5 using an Eppendorf Thermomixer R set at 30°C and mixing at 1400 RPM. Cells were subsequently back-diluted to an OD600 of 0.1 in SC + 2% glucose + 1 μM beta-estradiol and 5-Ph-IAA (or equivalent volume of DMSO, the 5-Ph-IAA solvent) was added to a final concentration of 5 μM. Cells were then grown for 1–3 hours using a thermomixer before being immediately fixed in ice-cold 4% paraformaldehyde (PFA) in PBS pH 7.4 for 15 minutes. Fixed cells were imaged with a Nikon TI inverted microscope using a 100x oil-immersion objective (1.45 NA), a Yokogawa dual spinning disk confocal unit and a Hamamatsu ImagEM EM-CCD camera. Images were acquired using MetaMorph software. A 488 nm laser was used for imaging GFP and a 594 nm laser for mCherry fluorescent protein. 0.25-micron slices with a total depth of 8 microns were used for Z stack acquisition. Maximum intensity z-projections were used to generate the final images from collected Z stacks.

Images were acquired using a Nikon TI inverted microscope equipped with a Yokogawa CSU-10 dual spinning disk confocal unit, 16.0 μm-pixel Hamamatsu ImagEM EM-CCD camera, Nikon 100x NA 1.45 TIRF objective, and MetaMorph software. 488 and 594 nm lasers were used with 525/45 nm (GFP) and 609/57 nm (RFP) bandpass filters for imaging of green (mNeonGreen) and red (mNeptune) fluorophores, respectively, with exposure times of 122.12 ms. Z-stacks were collected in 0.2 μm slices with a minimum of 8 μm total depth. The two-dimensional images shown were flattened by maximum intensity z-projection. Microscopy was performed at room temperature.