Aim software for the lsm 510 meta

Images were acquired using a Carl Zeiss LSM-510 Confocor 3 system with 100 × 1.45 NA Plan-Apochromat objective and a pinhole of one airy unit. The system was driven by Carl Zeiss AIM software for the LSM 510 meta. 405/488/561 nm laser was used to excite Calcofluor-white (CFW)/GFP/RFPs, and emission was collected through the appropriate filters onto the single photon avalanche photodiodes on the Confocor 3. All CFW images were acquired through a 420 nm long pass filter, GFP images were acquired through a 505–540 nm filter, and RFP images were acquired with a 580–610 nm filter on the Confocor 3. All images were acquired in a multi-track, alternating excitation configuration so as to avoid bleed through. GFP-fusion strains were imaged with excitation laser at 8 kW/cm². Any strains used in comparison were acquired with the same laser and scanning setting to compare their expression levels. All image processing was performed in the Image J software (NIH, Bethesda, MD). The total amount of each protein (e.g. Tom70-GFP) or fluorescent dye (e.g. TMRM) was quantified from the sum projection of Z-stacks after extracting the background signals. For visualization purposes, images scaled with bilinear interpolation were used for figures.