Csu10 spinning

Cells were also imaged and manipulated using an Olympus IX71 inverted microscope with a Yokogawa CSU10 spinning-disc scan head (Yokogawa Electric Corporation) equipped with a fast piezo objective z-positioner (PIFOC, Physik Instrumente GmbH & K.G.) and an Olympus UPlanSApo × 100/1.4 NA oil objective (Olympus). For cells expressing GFP and tdTomato/mCherry, we performed sequential imaging and images were acquired at a 2.1–4.7 s time intervals. For excitation, a sapphire 488 nm solid-state laser (75 mW; Coherent) and a Jive 561 nm solid-state laser (75 mW; Cobolt) were used for GFP and tdTomato/mCherry, respectively. The laser intensity was controlled using the acousto-optic tunable filter inside the Andor Revolution Laser Combiner (ALC, Andor Technology). The emission wavelength was selected using respective emission filters BL 525/30 (Semrock) and ET 605/70 (Chroma) mounted in a fast, motorized filter wheel (Lambda 10B, Sutter Instrument Company). The images have a xy-pixel size of 168 nm and the z-distance between optical sections was 500 nm. The system was controlled by Andor iQ software version 1.9.1 (Andor Technology).

FRAP experiments were performed using a FRAP photoablation module with a computer-controlled fiber optically pumped dye laser to bleach a region of interest (ROI) (∼2 μm in diameter) on the condensate or the centrosome after a few pre-bleach images were acquired. After photobleaching, the same ROI continued to be imaged at 2- to 5-s intervals for 5 to 12 min. Images were acquired using a Zeiss AxioObserver with a 60× objective coupled with a Yokogawa CSU-10 spinning disk confocal system anda Photometrics CoolSNAP HQ2 cooled charged-coupled device camera (BioImaging Solutions, San Diego, CA, USA). The microscope system was enclosed in an environmental chamber with the temperature set at 37°C. The photoablation and image acquisition were controlled by SlideBook software (Intelligent Imaging Innovations, Denver, CO, USA). Images and data were analyzed using ImageJ, Microsoft Excel and GraphPad Prism (GraphPad).