Deltavision mpx microscope

Cells were grown to mid-log phase in complete synthetic dextrose (SD) medium or SD–uracil (for selection for mitochondrial blue fluorescent protein plasmid) or SD–uracil–leucine (for selection of mitochondrial blue fluorescent protein and the Vps13^GFP plasmids). Images were acquired using a DeltaVision MPX microscope (Applied Precision) equipped with a 100× 1.40 NA oil UplanS-Apo objective lens (Olympus), a multicolor illumination light source, and a CoolSNAPHQ2 camera (Roper Scientific). Image acquisition was done at RT. Images were deconvolved with SoftWoRx software using the manufacturer’s parameters. Images were processed further using FIJI ImageJ bundle and assembled on Adobe Illustrator CS6. A single Z-section is depicted in the figures unless otherwise mentioned. For vacuolar staining, cells were pulsed with FM4-64 (Molecular Probes) at a concentration of 5 µg/ml for 20 min in the dark at 30°C. After washing, cells were chased for another 20 min in medium without FM4-64 and imaged subsequently. Quantification of colocalization was performed automatically in ImageJ (see the scripts in the online supplemental material).

Optical z-sections of live yeast cells were acquired with a Zeiss Axio Imager M2 equipped with a Zeiss Axiocam 506 monochromatic camera, 100× oil-immersion objective (Plan-Apo, NA 1.4), a Zeiss LSM800 equipped with an Airyscan detector, 63× oil-immersion objective (Plan-Apo, NA 1.4) or a Zeiss LSM880 equipped with an Airyscan detector, 63× oil-immersion objective (Plan-Apo, NA 1.4). Widefield images were acquired with ZEN (Carl Zeiss) and processed with Fiji (Schindelin et al., 2012 (link)). Superresolution images were acquired with ZEN (Carl Zeiss) and processed using the automated Airyscan processing algorithm in ZEN (Carl Zeiss) and Fiji. Individual channels of all images were minimally adjusted in Fiji to match the fluorescence intensities between channels for better visualization. Line scan analysis was performed on nonadjusted, single z-sections in Fiji. For Fig. S1 E, yeast were imaged on a DeltaVision MPX microscope (Applied Precision) equipped with a 60× 1.42 NA oil Plan-ApoN or a 100× 1.40 NA oil UplanS-Apo objective lens (Olympus), a multicolor solid state illumination light source, and a CoolSNAP HQ2 camera (Roper Scientific). Acquisition and deconvolution were performed with SoftWoRx software.