Fluoview fv3000 scanning confocal microscope

Time-lapse confocal imaging of nuclear import was performed on an Olympus FLUOVIEW FV3000 scanning confocal microscope, using a 40x air objective (NA = 0.95). An automated multi-position acquisition was carried out, where 12 different regions (typically containing 10 cells each) were imaged in two different wells. Three channels were recorded at each time step, using the 405 nm (Hoechst), 488 nm (Mitotracker) and 640 nm (cargo) laser lines for excitation. Images were acquired every 2 min for 80–90 min, using continuous autofocusing with Z-drift compensation to ensure imaging stability.

Confocal z-stacks were collected using a A1R MP+ confocal scanhead mounted on an Ni-E upright microscope (Nikon) using a 16× water dipping objective (NA 0.8) for live imaging or 40× oil immersion objective (NA 1.3) for fixed image acquisition. Images acquired in resonant scanning mode were post-processed using the denoise.ai function in NIS-Elements (Nikon). For live imaging, zebrafish were anesthetized in a solution of 0.006–0.012%buffered MS-222 in system water for 5 min. Anesthetized fish were mounted in a custom imaging chamber, partially embedded in 1% agarose, and covered with MS-222-containing solution. For Video 1, a FLUOVIEW FV3000 scanning confocal microscope (Olympus) equipped with a 100× objective (NA 1.49) was used to collect a z-stack which was 3D rendered with Imaris (Bitplane).