Perspective drawing of the microfluidic system used for time-lapse microscopy. The top transparent acrylic layer (AL) and the bottom perforated metal layer (ML) support the system assembly by means of eight thumbscrews (TS). In the middle from above, the hexa-device (HD) made of silicone elastomer micropatterned with two serpentine channels, connected to inlet and outlet channels (IN and OUT) for solutions to be perfused by means of a dual syringe pump. The solution (green) crosses the permeable membrane (PM), on which bacterial cells (BC) are seeded in superimposition with the 6 widest areas of the serpentine, and covered by a glass coverslip (GC), to enable monolayer growth. Bacteria are observed through the lens of an inverted microscope that can travel across the entire open area (OA) of the ML. Representative time-lapse image series of exponentially growing M. smegmatis P2P1recA-GFPwt (C) and P2P1recA-GFPdes (D) reporters. Phase-contrast and green fluorescence are merged. Numbers represent hours. Scale bar, 10 µm. Histograms showing the distributions of single-cell fluorescence (n = 204) averaged over the lifetime of the cell (right panels). Black line indicates fitting of the data with a Lognormal function (C) or with the sum of three Gaussians (D). Insets: P2P1recA-GFPwt population's geometric mean (Gµ) calculated by computing the logarithm of the single-cell fluorescence values, the mean of the logarithms and its antilog, P2P1recA-GFPdes subpopulations' means (µ), and R2 values. The two distributions show that the destabilized GFPdes is more suitable to monitor the dynamics of recA expression and to detect subpopulations, which are masked by using the stable GFPwt variant. Spearman's correlation between single-cell P2P1recA-GFPdes fluorescence range and VMR of fluorescence over the lifetime of the cell (n = 204). The data shown are from 3 independent experiments. Dashed lines separate different levels of fluorescence dispersion over the lifetimes of single-cells. Colors denote cells belonging to underdispersed (gray), dispersed (dark green), and highly dispersed (light green) subpopulations. P2P1recA-GFPdes fluorescence range over the lifetime of the cell of subpopulations segregated by fluorescence VMR (40 < n < 112). The data shown are from 3 independent experiments. Black lines indicate means ± SD. Asterisks denote significant difference by Kruskal-Wallis and Dunn's multiple comparison test: ****p < 0.0001. Single-cell time traces of P2P1recA-GFPdes fluorescence expressed as a percent of generation time of subpopulations segregated by fluorescence VMR. The average fluorescence (solid black lines) and half of the generation time (dashed lines) are indicated. Population statistics (n = 204): 38% (0<VMR≤1); 44% (1<VMR≤10); 18% (VMR>10). The data shown are from 3 independent experiments. Histogram showing the distribution of the fluorescence peak time expressed as a percent of generation time. Dark green bars indicate moderately pulsing cells (n = 165). Light green bars indicate highly pulsing cells (n = 66). Significance by Mann-Whitney test. The data shown are from 3 independent experiments. Peaks mainly occur between 40% and 70% of the generation time.