C13440 camera

Cells were prepared for imaging as described above. Cells were placed in an environmental chamber at 37°C with 5% CO₂ attached to an Axio Observer Z1 spinning disc confocal microscope (Zeiss). Micro‐irradiation was performed using a single‐point scanning device (UGA‐42 Firefly, Rapp OptoElectronic). Single‐point regions of interest (ROI) were defined for each cell and irradiated with 100% 405‐nm laser power for 600 iterations after removal of the ND filter. The estimated power delivered per ROI on average was approximately 27 J/m². ROI x–y co‐ordinates were recorded and used for subsequent image analysis. A predefined imaging template was used within the Zen Blue acquisition software. A pre‐irradiation image was recorded, followed by 405‐nm irradiation. A time lapse was subsequently performed every 5 s for 10 min. Hardware autofocus (Definite Focus, Zeiss) was used to ensure focus was maintained throughout the time lapse and was applied every 70 frames. To avoid image acquisition during laser micro‐irradiation, a 3‐s delay was applied from the start of micro‐irradiation and the beginning of the time lapse. Images were acquired using a C13440 camera (Hamamatsu), using a C Plan APO 64×/1.40 oil objective, acquiring 4× 0.5 µm optical sections per image with 4 × 4 binning.