Single-cell quantification of GFP-tagged protein levels reveals diverse proteomic changes during nitrogen starvation. (a) To track single cells, we integrated a cassette for expressing cytosolic mCherry into every strain of the yeast GFP collection. (b) We then automatically acquired images of hundreds of individual cells from each strain during growth in control conditions (logarithmic growth in SD) and three different stress stimuli. Using the mCherry (1), we segmented images (2) into single cell objects (3) and extracted GFP intensity in a single-cell resolution (4). Manual inspection of the library enabled the determination of subcellular localization for each protein (bottom). Bars, 5 µm. (c) A scatter plot (r² = 0.97) of median fluorescence values measured for each of the 5,330 strains of the yeast GFP library in two independent experiments in SD (a.u., arbitrary units). (d) Bubble plot depicting relative abundance (in SD) indicated by the size of the circle and sorted by the log₂ ratio of all measured strains under nitrogen starvation relative to SD. Red represents up-regulated events, blue represents down-regulated events, and black circles mark strains that were found to be significantly nonunimodal (i.e., to exhibit two subpopulations) by a Hartigan’s dip test. (e) Single-cell resolution analysis reveals bimodal distribution of expression of ribosome particles and ribosome-associated chaperones during nitrogen starvation relative to SD. Insets shows mCherry expression to remain unimodal in each strain under the same condition. This experiment was performed once on at least 100 cells per sample.