High-Content Microscopy of Protein Aggregation

Microscopy was done using a PerkinElmer Operetta microscope as established previously^{9 (link)}. Four sites were acquired per well. Laser-based autofocus was performed at each imaging position. Images of two channels (DAPI and GFP) were collected using a 60 × high-NA objective to visualize the cell and the aggregation states of the proteins, respectively. At every site and every fluorescent channel 5 images were taken at different z positions with 0.5 μm shifts. These images were used for a perfect focus algorithm. Cellular properties of about 1000 cells of each expressing strain were extracted from the images, including the localization of the GFP signal within the cell.