Trf89902 em

Fluorescence microscopy used an inverted Olympus IX83 microscope equipped with a 100× oil-immersion TIRF objective (numerical aperture, NA = 1.49) or (for Fig. S1) a 60× oil-immersion objective (NA = 1.45). Illumination for TIRF microscopy was via a Cairn MultiLine LaserBank (488 and 647 nm) and an iLas2-targeted laser illumination system (Cairn Research), through which the excitation angle for TIRF was adjusted to achieve a theoretical penetration depth of 80 to 100 nm. Excitation light of 488 or 647 nm was passed through a quad-band filter set (TRF89902-EM, Chroma Technology), and emitted light was passed through an appropriate band-pass filter within a high-speed filter wheel (Cairn Optospin; peak/bandwidth: 525/50 or 700/75 nm). A 395-nm LED (SPECTRA X-light engine, Lumencor) was used for flash photolysis of ci-IP₃. Detection of emitted light was via an EMCCD camera (iXon Ultra 897, Andor; 512 × 512 pixels; pixel size = 0.16 μm at 100× magnification). Image capture used MetaMorph Microscopy Automation and Image Analysis Software (version 7.10.1.161, Molecular Devices), through which image capture (control of shutters, etc.) was fully automated. Both live and fixed cells were imaged at 20 °C.

DNA-PAINT microscopy was performed on a home-built SMLM setup with an Olympus IX81 inverted microscope frame equipped with an Olympus 150× TIRF oil immersion objective (UIS2, 1.49NA). The samples were illuminated in HILO mode^{40 (link)} using a 561 nm laser line (Coherent Sapphire LP) at an illumination density of 0.88 kW/cm² through a 4 L TIRF filter (TRF89902-EM, Chroma Technology) and ET605/70 M nm bandpass filter (Chroma Technology). Signals were detected with an Andor iXon EM+ DU-897 EMCCD camera (Andor, Ireland). SMLM frames were acquired using multi-dimensional acquisition (MDA) mode in Micro-Manager 2.0-gamma^{41 (link)}.