Live-cell HILO imaging was performed on a custom total internal reflection fluorescence (TIRF) microscope (assembled by CAIRN Research). The system is based on an Eclipse Ti2 microscope (Nikon, Japan) equipped with four EMCCD cameras (iXon Ultra 897, Andor), 100x oil-immersion objective (SR HP APO TIRF NA 1.49, Nikon), an iLas2 TIRF illuminator (Gataca Systems), 405, 488, 561, and 637 nm diode lasers (Coherent, Obis), a quadruple beam splitter, quadruple band excitation and dichroic filters, 1.5x tube lens and hardware focus stabilization. Two of the four synchronized EMCCSs were used to acquire simultaneous image sequences at a rate of one image every 30 s. HILO images were acquired with MetaMorph 7.10.2.240.