Plan apochromat 20 0.8 na m27 objective

The inverted confocal microscope system (Zeiss LSM 880 and HiS‐SIM) was adopted for live imaging. For Zeiss LSM 880, the microscope was mounted on a Zeiss Axio Observer Z1 basic stand equipped with an incubator (XLmulti S1) to maintain the temperature at 37 °C and 5% CO₂. The Plan‐Apochromat 20×/0.8 NA M27 objective (Zeiss) was used for imaging. Time‐lapse images were acquired every 10 s for 30 min. The laser excitation and filter were set for enhanced GFP using the settings detailed above. Image processing was performed with ZEN software (blue edition, Zeiss). HiS‐SIM (High Sensitivity Structured Illumination Microscope) is provided by the Guangzhou Computational Super‐resolution Biotech Co., Ltd.

Microscope-based PercevalHR ATP/ADP level detection was performed by using laser confocal microscopy. Briefly, 2 × 10⁴ cells were seeded in a 96-well clear bottom black plate (Costar, 3603). Images were taken by a Zeiss LSM880 confocal microscope using a Plan-Apochromat 20×/0.8 N.A. M27 objective at 37 °C. PercevalHR was excited using a 488 and 405 nm laser, for the MgATP-bound conformation and ADP-bound conformation, respectively. Emission was captured through a 450–550 nm filter. pHRed was excited using a 488- and 405-nm laser, and emission was captured through a 560 nm long pass filter. Four channel image sets were taken with no delay between individual channel acquisitions. For time-lapse studies involving PercevalHR, cells were imaged on IN Cell Analyzer 2500 HS high content analysis (HCA) imaging system using a 20×/0.8 N.A. objective. Time-series images were processed using Fiji. The investigators were blinded to sample allocation.
For flow cytometry, cells were collected and resuspended in HBSS. Ratios of FITC and KO525 channels of the CytoFLEX Platform (Beckman Coulter) were used to detect ATP/ADP levels. PH-correction was performed by using a BCECF-AM pH probe (DOJINDO, B262) according to the protocol provided by the producer.