Fig. 3

Single-cell ATP measurements in the hydrogel plugs carrying yeast cells that express QUEEN-2m, a genetically-encoded ratiometric GFP-based ATP biosensor. A Alphafold-predicted structure of QUEEN-2m, with annotated ATP-binding domain and circularly-permuted enhanced green fluorescent protein (cpEGFP). B Excitation spectrum of QUEEN-2m at different ATP concentrations. C Ratio of emissions at 520 nm following excitations at 410 and 480 nm, with orange shading marking the physiologically relevant ATP range and error bars denoting 95% confidence intervals. D Inner cross-sections of hydrogel plugs after overnight incubation in 1.5 ml of deionized water or media, with graphs showing 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 Control ATP-depleted cells (incubated in 2-D-deoxyglucose [50]) and ATP-rich cells (incubated in D-glucose), with purple color denoting low ATP content and yellow color high ATP content. Scale bars: 1 mm in D, 100 µm in E, and 20 µm in F, G, and H