Borealis beam conditioning unit

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.

All imaging experiments with the exception of those depicted in Fig 1B–1E and Fig B in S1 Fig were performed at 30°C on a Nikon Eclipse Ti inverted microscope equipped with a motorized laser total internal reflection fluorescence (TIRF) illumination unit, a Borealis beam conditioning unit (Andor), a CSU-W1 Yokugawa spinning disk (Andor; Belfast, Northern Ireland), a 60X PlanApo TIRF 1.49 numerical aperture (NA) objective (Nikon; Toyko, Japan), an iXon Ultra EMCCD camera (Andor), and a laser merge module (LMM5, Spectral Applied Research; Exton, PA) equipped with 405, 440, 488, and 561-nm laser lines. All hardware was controlled using Micro-Manager (UCSF). For experiments performed under confinement, the PDMS devices were connected with PE 20 tubing (Braintree Scientific; Braintree, MA) to a vacuum regulator (IRV1000-01B, SMC Pneumatics; Tokyo, Japan), which, in turn, was connected to the building-wide vacuum supply.
Imaging experiments depicted in Fig 1B–1E and Fig B in S1 Fig were performed at 37°C on a Nikon Eclipse Ti inverted microscope equipped with a motorized stage (ASI), a Lamba XL Broad Spectrum Light Source (Sutter; Novato, CA), 20x 0.75 NA Plan Apo and 60x 1.4 NA Plan Apo objectives (Nikon), and a Clara interline CCD camera (Andor). All hardware was controlled using Nikon Elements.