(A) Representative images of the same cell expressing ratiometric pHluorin imaged at the start of the experiment and after 80 hr; the replicative age is indicated. Young cells are trapped in the microfluidic device, and bright-field images are taken every 20 min to determine the age of the cells. Fluorescent images are taken once every 10 hr (panels, C, D, F) or every hour (panel E) with excitation at 390 and 475 nm and emission at 525 nm. The images show DIC, DAPI, and FITC channels and merged fluorescence. The scale bar represents 5 μm. The trapped aging cell is indicated with white arrowheads. (B) Calibration curve showing the relationship between intracellular pH and pHluorin ratios. Cells expressing pHluorin from an exponential culture at OD600 of 0.5 were resuspended in buffers, titrated to pH 5, 5.5, 6, 6.5, 7, 7.5, 8, containing final concentrations of 75 μM monensin, 10 μM nigericin, 10 mM 2-deoxyglucose, and 10 mM NaN3. Monensin and nigericin are ionophores that carry protons across the plasma membrane, while the 2-deoxyglucose and NaN3 deprive the cell from ATP and thus block energy-dependent pH maintenance. Each point represents data from 20 cells. (C) Data collected from 80 yeast cells during the process of replicative aging. The young group consists of data points from the first recorded ratio at time 0 hr, and the old group consists of the last recorded measurement before cell death. Colored crosses indicate the average and bold lines show the median, p=9.1E-18. (D) Single-cell profiles of the pH at different replicative ages. Cells are grouped according to lifespan (RLS <15, 15–20, 20–25, 25–30 and >30). Grey bar indicates the range of pH values measured in the first time point (young age) to illustrate that 36% of the cells remain within this range throughout their lifespan. (E) Cytosolic acidity (normalized ratiometric pHluorin read-out:F475/F390, top) and replicative age (bottom) as a function of time, for a cell that enters senescence at 26 hr (dashed blue line) after which a sharp acidification of the cytosol (dashed red line) follows. (F) The pH of single cells during aging plotted versus their age in hours. Each grey line represents a single cell. The orange line shows the average pH. The dark grey circles show the total proteome pI in aging estimated based on the PI and changes in abundance of 1229 proteins. Error bars represent the standard error of the mean (SEM).