Hardware used for the experiments included an Eclipse Ti inverted microscope equipped with a motorized laser TIRF illumination unit, a Borealis beam-conditioning unit (Andor Technology), a 60× Plan Apochromat TIRF 1.49 NA objective (Nikon), an iXon Ultra electron-multiplying charge-coupled device camera, and a laser merge module (LMM5; Spectral Applied Research) equipped with 405-, 488-, 561-, and 640-nm laser lines. All hardware was controlled using Micro-Manager [36 ,38 ] (University of California, San Francisco), and all experiments were performed at 37°C and 5%CO2.
Activity of opto-PI3K was controlled via a 470-nm (blue) LED (Lightspeed Technologies) that transmitted light through a custom DMD (Andor Technology) at varying intensities by connecting the LEDs to the analog outputs of a digital-to-analogue converter and setting the LED voltages using serial commands via custom Python code. Our microscope is equipped with two stacked dichroic turrets such that samples can be simultaneously illuminated with LEDs using a 488-nm longpass dichroic filter (Chroma Technology) in the upper turret while also placing the appropriate dichroic (Chroma Technology) in the lower turret for TIRF microscopy.
Activity of opto-PI3K was controlled via a 470-nm (blue) LED (Lightspeed Technologies) that transmitted light through a custom DMD (Andor Technology) at varying intensities by connecting the LEDs to the analog outputs of a digital-to-analogue converter and setting the LED voltages using serial commands via custom Python code. Our microscope is equipped with two stacked dichroic turrets such that samples can be simultaneously illuminated with LEDs using a 488-nm longpass dichroic filter (Chroma Technology) in the upper turret while also placing the appropriate dichroic (Chroma Technology) in the lower turret for TIRF microscopy.