Perfect focus 2

Fluorescent imaging was performed at room temperature using a Yokogawa CSU W1 spinning disk attached to a Nikon Ti2 eclipse with a Photometrics Prime 95B camera using Nikon Elements software (versions 5.20.00 to 5.21.02). Imaging was performed through a Nikon Plan Apo 60× 1.40 NA oil objective with Immersol 518 F (Zeiss; refractive index 1.518), and Perfect Focus 2.0 (Nikon) was engaged for all captures. For single time point captures using the Ibidi Angiogenesis slides, images were taken at the surface of the sample at 30 min after induction or at the indicated time point. Imaging was performed using 488-nm, 555-nm, and 640-nm lasers when applicable along with a capture of DIC. Multipoint time-lapse imaging was performed in Lab-Tek chambered cover glass slides, where 5 xy fields were stored for each condition. Images were taken at each xy position every 10 min. Automated granule detection and measurement were performed using CellProfiler software similar to the methods described previously (Mackenzie et al., 2017 (link)), excluding association of granules to cells. Briefly, the lysate granules were segmented by applying a global minimum cross entropy approach on the GFP channel. The sizes represent an average of at least five separate fields, with each field containing at least 100 segmented granules.