Cfi plan apo 1.4 na

Images were acquired on a Nikon a1r inverted confocal microscope with a ×60 objective (oil; CFI Plan APO 1.4 NA) and typically one image was acquired every 1 s. Rapid ratiometric changes of fluorescence measured with Cox8‐pHred were recorded using two excitation wavelengths (405 and 561 nm) and one 624/40 emission filter. Time‐resolved pH and potential imaging was performed at 37°C on cells transiently transfected with mito‐sypHer or pHluorins and loaded with 4 or 20 nM TMRM for HeLa and MEF cells, respectively. Images were acquired using 488‐ and 561‐nm excitations and 520/35 and 624/40 emission filters. The frequency of depolarizations and pH flashes was analyzed using ImageJ.

WT MEFs and Opa1^−/− cells transfected with 6 μg pCXN2‐OPA1 or OPA1^K301A (gift from Prof. Yoshihiro Kubo) and 1 μg mito‐PA‐GFP and 0.5 μg mito‐DsRed were imaged in DMEM media. Cells with low GFP fluorescence intensity were selected to avoid saturation of the GFP emission upon photoactivation 62. GFP and mito‐DsRed were imaged concurrently on a Nikon A1R confocal microscope with a × 60 objective (oil; CFI Plan APO 1.4 NA) using 488 and 561 nm excitation and 520/35 and 624/40 emission filters. Three images were acquired (one image/second) before applying three stimulation pulses (500 ms, 405 nm laser 50 mW, 7% power) followed by live imaging. Loss of focus was minimized by using the Perfect Focus system (Nikon). ImageJ was used for data analysis of the area of PA‐GFP. Fusion was quantified as the % of PA‐GFP area increase after 60 min compared to the initial PA‐GFP area measured 1 min post‐photoactivation in the same cell.