Plan apochromat 40 1.4 oil objective lens

Cells were imaged on a Zeiss Elyra 7 system using a Plan-Apochromat 40×/1.4 oil objective lens. Multicolored acquisition was performed sequentially to minimize cross-talk between channels. The fluorescent images were capture on two PCO Edge sCMOS cameras attached to a DuoLink motorized dual camera adapter and the color split using the secondary beam splitter BP420-480 + BP470-640 + LP740. The fluorescent protein mCherry was excited using the 561 nm laser line and emission collected from 570 to 640 nm. The fluorescent protein GFP was excited using the 488 nm laser line and emission collected from 490 to 570 nm. Apotome acquisition was set to collect five phase images with 25 ms camera exposure time. Yeast cells were optical Z sections using step size optimized for “Leap” acquisition. For time-lapse experiments z stacks were collected with an interval of 4.3 s. Apotome phase images were processed using Zeiss Zen Black software set to 3D SIM Leap. The alignment between the color channels was further improved by taking a Z stack of multicolor TetraSpec microspheres (ThermoFisher) that was used to generate an alignment matrix using the “Channel alignment” tool in Zen Black and applied to the time-lapse data.