Proteins carry out most of the functions of the cell. Therefore, dissecting how proteins are organized is fundamental to our understanding of biological processes. We present a microscopy setup that we have been developing to quantify protein concentrations and distributions in living yeast cells at high-throughput. The live cell imaging is performed using a spinning disk confocal microscope. It enables the imaging of a 384 well-plate with three channels and in 3D in under an hour. The resulting images are processed automatically, enabling the analysis of thousands of images. As each image typically contains hundreds of cells, this setup uniquely allows investigating phenotypic variability. We have been developing ImageJ plugins to allow cell segmentation from brightfield images and the extraction of fluorescence properties from individual cells. We present three applications of these plugins. In a first use, we examined cell-to-cell variability in protein concentration and the impact that protein degradation can have on this variability. In a second implementation, we examined the concentration dependence of protein aggregation. In a third project, we explored how protein concentration and stoichiometry impact the assembly of multivalent interacting proteins.
