Flash 4.0 camera

Lens imaging was performed as previously described ⁷⁵ . Briefly, whole intact eyes were dissected from live, anesthetized fish larvae and examined under DIC illumination on Zeiss AxioImager.Z1 equipped with 20X/0.80 Plan Apochromat objective and Flash4.0 camera (Hamamatsu). For transmitted light imaging, lens was further dissected from eye and image was acquired by AxioCam HRc (Zeiss) camera on AxioImager.Z1equipped with 20X/0.80 Plan Apochromat objective.

Microscopy images were collected on an inverted widefield fluorescence Nikon Ti-E microscope equipped with a Hamamatsu Flash4.0 camera using PlanApo 20x or 40x air objectives. Samples were imaged in a Greiner Bio-One 384-well glass bottom plate PEGylated using 20 mg/mL PEG-Silane (Laysan Bio, MPEG-SIL-5000) and passivated with 100 mg/mL BSA as described^{51 (link)}. Prior to addition of samples, the wells were washed 3x with 25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT. Dcp1/Dcp2 constructs assayed for phase separation by microscopy were prepared by initiating removal of the N-terminal MBP solubility tag with 1:40 molar equivalent TEV:Dcp1/Dcp2. Dcp1/Dcp2/Edc3 droplets were prepared by incubating Dcp1/Dcp2 and Edc3 prior to removal of the N-terminal MBP tag from Dcp1/Dcp2_ext. Imaging was performed after 30 minutes to ensure TEV cleavage and droplet. Image analysis was performed using ImageJ^{52 (link)}. For localization and enrichment of Dcp1/Dcp2_ext, Edc3, or RNA in droplets, 1%–5% protein concentration was fluorescently labelled. Enrichment was estimated from the average ratio of intensity in at least twenty droplets (I_droplet) to average intensity in surrounding solution (I_dilute).

Brightfield images were collected on a Nikon Ti-E inverted microscope equipped with a PlanApo 40x air objective and Hamamatsu Flash4.0 camera. For wild-type Dcp1/Dcp2_ext, time lapse images were collected every one second while images for Dcp1/Dcp2_ext(Y220G) were collected at either one second or 15 second intervals. Fusion events were analyzed in ImageJ by drawing an ellipse around droplets of similar size at each time point and measuring the long and short axis. The aspect ratio (long/short axis) was plotted as a function of time in Prism 9 (GraphPad) to determine the time of fusion, τ. Plotting τ as a function of initial length ((l_long – l_short)•l_short)^1/2 gives a linear relationship where the slope represents the inverse capillary action—the ratio of condensate viscosity to surface tension (η/γ)^{59 (link)}.

Cells were imaged in epifluorescence after fixation with 4% paraformaldehyde (Electron Microscopy Sciences) solution in PBS and subsequent washes with PBS. Images were acquired on a Deltavision system (Applied Precision) equipped with a 60X 1.42 numerical aperture oil objective and SoftWoRx software (Applied Precision). Deconvolution of fluorescence images using measured point-spread function was done in the SoftWoRx software, and final images were processed using FiJi^{32 (link)} or Metamorph software.
All total internal reflection fluorescence microscopy experiments were performed on a custom-built microscope based on an Olympus IX-70 microscope, equipped with a UApoN 100× 1.49 NA oil objective, a 488-nm, 594-nm, and a 647-nm laser. The cells were imaged under azimuthal TIR-FM^{33 (link)} using a Cairn Opto-Split III with a cube and appropriate filters and a Hamamatsu Flash 4.0 camera. Multiple colors were aligned by using calibration data obtained with 500 nm fluorescent beads (TetraSpeck Fluorescent Microspheres Size Kit, T14792, Invitrogen) mounted on a slide^{34 (link)}. The microscope was pre-warmed to 37ºC prior to imaging and temperature was maintained during imaging.