Conventional TIRFM was performed on an Olympus cellTIRF-4Line system using a 150× (NA 1.45) oil objective. Confocal images were acquired using a Zeiss 780 LSM using a 40× water objective (NA 1.2). Imaging of live samples was performed at 37°C, and of fixed samples at room temperature. Super-resolution imaging was performed on a custom-built TIRF-SIM setup based on a ferroelectric spatial light modulator used to generate diffraction patterns and adjust the TIRF angle (Li et al., 2015 (link)). The TIRF angle was selected to ensure below 150 nm penetration depth 488, 560, and 640 nm laser lines. Illumination and detection was performed through an Olympus 100× (NA 1.49; UPLAPO100XOHR) oil objective. Raw images were obtained on two Hamamatsu Orca Flash 4.0 cameras, and reconstructed with custom made software (Li et al., 2015 (link)). Multi-channel TIRF-SIM images were corrected for chromatic aberrations using the MultiStackReg plugin for ImageJ and 0.1 μm TetraSpeck microspheres (ThermoFisher Scientific) on glass as a reference standard.
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