35 mm dish

After droplet formation, an aliquot of the reaction mixture (200 μM 25QP-GFP in 10% dextran, 150 mM NaCl, 50 mM Na₂HPO₄/NaH₂PO₄, pH 7.4) was added to a glass-bottomed dish (35mm dish, 14mm glass diameter; MatTek) that had been treated with 30 mg/mL BSA (Sigma) for 30 mins. The dish was sealed to prevent evaporation and the droplets were imaged by time-lapse fluorescence microscopy using the custom-built TIRF microscope described above (Total Internal Reflection Fluorescence (TIRF) Microscopy), set up in a configuration with the fast steering mirror angled to improve signal-to-noise ratio. Images were collected every 10 s for 1 – 2 hr until the droplets had converted. The laser was operated at 0.1% power (∼0.1mW) and was further reduced in power by placing a half-wave plate and polarizing beam splitter (Thorlabs) in the beam path. Experiments were carried out at room temperature (21°C).
The circularity of the droplets over time was measured by applying automatic local thresholding to each frame using the Otsu method (Otsu, 1979 ) in Fiji to create a binary image of the droplets, then measuring the areas and perimeters. CRs were calculated as described (Intensity and circularity ratios) in R and the mean CR in the field of view was plotted over time.

For each condition 1 × 10⁷ parasites were transiently transfected with 30 µg of pTub SAG1ΔGPI-mCherry plasmid (Heaslip et al., 2016 (link)) and immediately added to a confluent HFF monolayer in a Mattek 35 mm dish, coverslip 1.5. 12–15 hr after infection the monolayers were washed three times with pre-warmed Gibco Fluorobrite DMEM supplemented with 4% Fetal Bovine Serum. The coverslips were immediately used for imaging. The acquisitions were made with an Olympus IX71 coupled to a DELTAVISION Elite imaging system in a 37°C environmental chamber. The acquisition for each condition were made sequentially from the washing step to the acquisition with a random order to avoid any artifactual data. The acquisition analysis was made with Fiji and the MTrackJ plugin. The data represent three independent experiments. At least 42 parasites and 273 direct runs were counted per strain. The results were statistically tested with a one-way ANOVA test plus a multiple comparison Dunnett’s test comparing all means to the ΔCAP^CAP mean in GraphPad Prism 7. The data presented are as mean ± s.d.