Fig. 4

Single-cell measurements of cytosolic pH in the hydrogel plugs carrying yeast cells that express sfpHlourin, a genetically-encoded ratiometric GFP-based pH biosensor. A Alphafold-predicted structure of sfpHlourin, a superfolder green fluorescent protein (sfGFP) sensitive to pH changes. B Excitation spectrum of sfpHlourin at different pH. C Ratio of sfpHlourin emissions at 520 nm following excitations at 410 and 480 nm, with orange shading marking the physiologically relevant pH range and error bars denoting 95% confidence interval. D Inner cross-sections of the plugs incubated in 1.5 ml of water or media, with graphs denoting the mean ratiometric signal. E Edge details of the micrographs in D. F Superstructure in the periphery of the media-incubated plugs. G Microcolonies at the edge and in the center of water- and media-incubated plugs. H Permeabilized control cells incubated in pH 5.5 and pH 7.0, with purple color denoting lower cytosolic pH and yellow color higher cytosolic pH. Scale bars: 1 mm in D, 100 µm in E, and 20 µm in F, G, and H