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Pipeline publication

[…] goat anti–rabbit), and Alexa Fluor 633 (goat anti–mouse) were used at 1:1,000 dilution (Invitrogen). Images were acquired using an UltraVIEW VoX spinning disc (CSU-X1; Yokogawa Corporation of America) confocal microscope (PerkinElmer) on a microscope (Ti; Nikon) equipped with a 60× (Apochromat total internal reflection fluorescence 60× oil, NA 1.49) objective lens and camera (EMCCD C9100-50; CamLink). Live cells were imaged in DMEM medium at 37°C with 5% CO2. Standard image processing was performed using Volocity (PerkinElmer) and ImageJ (). No image manipulation was performed, except a background subtraction in (ImageJ, rolling ball with 10 pixel radius). Images in were deconvolved using Huygens Professional with the “Classical Maximum Likelihood Estimation (CMLE) algorithm” and measured point spread functions of 488-, 546-, and 634-nm channels (Scientific Volume Imaging) before manual analysis for colocalization. For colocalization analyses, S-OPA1 punctae near the periphery of the cell were scored for their 3D coincidence with DRP1, MID49, or ER–mitochondria contact sites using Imaris (Bitplane). 3D visualization in was performed in Volocity (PerkinElmer) using “3D opacity view.”, 5 × 104 cells were transfected with 1 µg matrix-PA-GFP and 0.5 µg mito-mCherry, and imaged after 24 h. Approximately 23 ± 12% of mitochondria were photoactivated using 5% power of a 405-nm laser for one cycle (30 iterations). Z stack images of PA-GFP and mito-mCherry using a step size of 0.3 µm were acquired using 488-nm and 568-nm lasers immediately after photoactivation (1 min delay) and after 20, 40, or 60 min. We thank O. Shirihai (Boston University, Boston, MA) for the plasmid encoding IM-PA-GFP and A. Kumar Kondadi (University of Cologne, Cologne, Germany) for the plasmid encoding mito-mCherry., Mitochondrial fusion was quantified using […]

Pipeline specifications

Software tools Huygens, Imaris, Volocity