Csu x1 spinning disk

Fluorescence
microscopy imaging of protein droplet formation was performed at ambient
temperature (approximately 22 °C) on an Olympus IX81 inverted
confocal microscope (Olympus Life Science; Tokyo, Japan) equipped
with a Yokogawa CSU-X1 spinning disk, mercury lamp, 488 and 561 nm
laser launches, iLas-targeted laser system for photobleaching and
an iXon3 EMCCD camera (Andor; Belfast, UK). Multidimensional acquisition
was controlled by MetaMorph software (Molecular Devices; Downingtown,
PA). Samples were illuminated using a 488 nm laser and imaged through
a 100×/1.4 NA oil-immersion objective. To image in vitro droplet
formation, proteins were thawed at 50 °C, diluted to 4 μM
in a buffer containing 150 mM NaCl and 20 mM Tris-HCl (pH 8.5 unless
otherwise specified), and placed on custom-fabricated acrylic gasket
chambers adhered to glass coverslips.

Strains were grown overnight in YPD to saturation, then diluted 20-fold into SC-Trp with β-estradiol to a final concentration of 2 μM for 1.5 hours. Yeast cells were mixed with low melting point agarose to limit motion while imaging and applied to a standard microscopy slide for live imaging using a Nikon Ti2-E equipped with a Yokogawa CSU-X1 spinning disk and a 100x NA = 1.49 oil objective. Samples were illuminated with a 488 nm solid state laser light source and images collected on an ORCA-FLASH 4.0 sCMOS camera. A single z-slice through the center of the cell was acquired for two fields of view in a single biological replicate. Image processing was performed using ImageJ (Collins, 2007 ; Schneider et al., 2012 (link)).

Embryos were dissected into M9 buffer and mounted on a 3% agarose pad and imaged on a spinning disk confocal microscope built around a Zeiss Axio Observer Z.1 equipped with a Zeiss Plan-Apochromat 63×/1.4NA oil immersion objective, a CSU-X1 spinning disk (Yokogawa), an Evolve 512 × 512 electron-multiplying charge-coupled device (EMCCD) camera (Photometrics) and a 50-mW 488-nm solid state laser and controlled by the Slidebook software package (3I, Inc). The still images of MEG-3::meGFP in Figures 1 and 6 were taken at the midplane with 60% laser power, 1-s exposures and camera intensification of 300 and gain of 1. Images of PGL-1::GFP in Figures 1 and 6 are from Z stacks (0.5-μm step size) covering the half of the embryo closest to the coverslip (the middle plane is shown in Figure 1, and maximum intensity projections are shown in Figure 6). Z stacks were collected using 20% laser power, 100-ms exposures, a gain setting of 1. To quantify the distribution of granules in Figure 6, granules with a diameter >0.7 μm were counted. For Supplemental Movie S4, images were captured at the middle plane at 15 s intervals with 20% laser power, 100-ms exposures, camera intensification of 300 and gain of 1 for the 488-nm channel and 60% laser power, 300-ms exposures, camera intensification of 300 and gain of 2 for the 640-nm channel.