Orca flash 4.0 v2

Images were acquired on a Nikon Ti-E system fitted with a Yokogawa CSU-X1 spinning disk head, Hamamatsu Orca Flash 4.0 v2 digital CMOS camera, Perfect Focus system, and a Nikon LU-N4 solid state laser launch (15 mW 405, 488, 561, and 647 nm) using a 100×1.49 NA Apo TIRF oil-immersion objective. This microscope was controlled through Nikon Elements AR software on a 64-bit HP Z440 workstation.

A Nikon-Microphot-SA microscope was used to capture images of DAB-stained embryos (40×). Images for the Sxl RNAi rescue experiment were acquired using a 100×, 1.49 NA Apo TIRF oil immersion objective on a Nikon Ti-E system fitted with a Yokagawa CSU-X1 spinning disk head, Hamamatsu Orca Flash 4.0 v2 digital CMOS camera, and Nikon LU-N4 solid state laser launch. Imaging for all other smFISH and fluorescent immunostaining experiments was performed on a Nikon A1 inverted laser-scanning confocal microscope.
Images were assembled using ImageJ (NIH) and Adobe Photoshop and Illustrator software to crop regions of interest, adjust brightness and contrast, generate maximum-intensity projections, and separate or merge channels. To assess the spreading of the RNAs or protein in different mutant backgrounds compared to the control we generated plot profiles using ImageJ. The posterior-most 75 µm of each embryo was plotted for comparison, and embryos from a single biological replicate are plotted in figures given that variation between fluorescence between replicates obscured the pole plasm distribution trends if embryos from all replicates were plotted together.

Phase separation of the B-LCD and the B-LCD–Δamy proteins was induced in 384-well plates (Matriplate, Brooks) by diluting stock solutions containing urea to the desired protein concentrations in 50 mM Tris, pH 7.5, 2 mM β-mercaptoethanol and different NaCl concentrations. Final urea concentrations were 200 mM or 400 mM.
Brightfield and widefield fluorescence microscopy were carried out on a Nikon Ti2 Eclipse inverted microscope, equipped with an LEDHub light source (Omicron) and an Andor Zyla camera, using a 60× oil objective (Nikon, numerical aperture 1.4). Image acquisition was controlled using the MicroManager software (version 2.0 gamma).
Confocal microscopy was performed with a Nikon NSTORM (Nikon) system equipped with a Rescan Confocal Microscope RCM1 (Confocal.nl). We used an sCMOS camera (Orca Flash 4.0 V2) and a Nikon SR Apochromat TIRF objective 100×/1.49 with oil immersion. Laser excitation wavelengths used were 488 nm, and 647 nm, to excite the ThT, atto488 and atto647 dyes, respectively. The set-up was fully controlled, and image acquisition was performed using the NIS Elements 5.20 Advanced research software (Nikon). The implemented re-scan unit provides an enhancement in resolution from 240 nm to 170 nm (refs. ^{76 (link),77 (link)}).
Droplet size distributions and numbers were extracted using an in-house written program in MATLAB (version R2020a).