Hcx pl apo 63x

Imaging was performed using an Andor Revolution spinning disk imaging system build on a Leica DMI6000B inverted microscope with adaptive focus control, with a Yokogawa CSU x1 spinning disk unit, and an Andor iXon EMCCD camera. The system is equipped with four solid state lasers at 445 nm, 488 nm, 514 nm, and 594 nm, combined through an acousto-optic tunable filter to enable rapid switching and control excitation power. An incubation chamber surrounding the entire microscope was maintained at 37°C, 5% CO₂. Imaging was performed using a 63x, 1.4NA oil immersion objective (Leica 506187: HCX PL APO 63x). For GFP-γ9 imaging and simultaneous activation of PPO, a single confocal plane was imaged at a rate of one frame every 3 s, with 488 nm excitation through the spinning disk (average power ~45 μW, which is typical for imaging GFP alone) and 300ms exposure time. PPO deactivation was achieved using a 595 nm LED (CoolLED pE-4000). The LED light was directed through the back port of the microscope by an Andor Mosaic DMD operating in “white mask” mode and ~150 μW measured through the objective. The 488nm imaging laser and the 595 nm LED were coupled into the excitation path using a 562nm long pass dichroic mirror (Semrock Brightline FF556-SDi01). GFP emission was collected through the same dichroic mirror, a 488nm notch mirror, and a 525/30nm emission filter.