Metabolic activity sensing under iron limitation at single cell level.

<p><b>(A)</b> Mean single cell fluorescence traces of all descendants of a progenitor cell are shown under intermittent iron supply. A daughter cell with higher calcein fluorescence than its siblings (light blue arrow) generated growing descendants with increased mean single cell fluorescence traces (light blue lines) in comparison to other descendants of the initial progenitor cell (grey mean single cell fluorescence traces). A single spontaneously non-growing cell changed from a dividing state to a non-growing state loosing esterase activity and intracellular calcein due to lysis (indicated by black arrow, lysed cell shown in <b>(C)</b> and <b>(D)</b>, respectively). <b>(B)</b> Mean single cell fluorescence traces of spontaneously non-growing cells of a microcolony are shown. Lysing cells (red lines) lost fluorescence spontaneously after lysis (red arrow). However, they were still detectable as apparently intact cells (end of recognition marked with red asterisks). The mean single cell fluorescence traces of a spontaneously non-growing but metabolically active cell (blue line) are shown in comparison. Mean single cell fluorescence increases shortly after cell birth (blue arrow). <b>(C)</b> A lysed but apparently intact cell (marked with black dashed line) and a cell directly before performing lysis (marked with red dashed line) are depicted. <b>(D)</b> Lysed cells still appear to be intact cells (red dashed line and black dashed line, respectively) after lysis. These non-growing cells showed no calcein fluorescence and were considered to be metabolically non-active. <b>(E)</b> A non-growing but metabolically active cell after re-supply of iron with elevated mean single cell fluorescence (marked by blue dashed line).</p

Metabolic activity sensing of cells exposed to unviable antibiotic concentrations and metabolic activity changes of descendants after antibiotic stress.

<p>After an initial growth phase in complex medium BHI, cells were exposed to <b>(A)</b> 10 μg/mL ampicillin (cell wall inhibition, n = 5 colonies) and <b>(B)</b> 10 μg/mL chloramphenicol (inhibition of protein synthesis, n = 5 colonies), respectively, for one hour. The antibiotics were added to the perfusion medium during the exposure time and after an hour perfusion with antibiotic free BHI was continued. Calcein mean single cell fluorescence revealed how the antibiotics change the bacterial fitness during antibiotic exposure. (<b>C</b>) The apparent growth rate was determined for five microcolonies treated for one hour with AMP or CHL.</p

Experimental validation of the metabolic activity sensing method.

<p><b>(A)</b> The CAM-surrogate methyl methoxyacetate was added to CGXII + 4% GLC and infused in three different concentrations in three separate supply channels of the microfluidic device to analyze the impact on growth by the intracellular digestion of the acetoxymethyl ester groups of CAM. The average maximal growth rates of five chambers cultivated with CGXII + 4% GLC with addition of three different methyl methoxyacetate concentrations are compared to a reference without addition (n = 5 colonies). The tested methyl methoxyacetate concentrations at 5 μM, 50 μM, and 500 μM corresponded to a CAM concentration of 1.7 μM, 16.7 μM, and 166.7 μM, respectively. In comparison the result for bacterial growth in CGXII + 4% GLC <b>+</b> 46 μM CAM is given. <b>(B)</b> An influence of the excitation light exposure (phototoxicity) during the fluorescence time lapse imaging was investigated by infusing DHCAM, which emits green fluorescence if light induced oxygen radical species are present. Typical experimental light exposure resulted in exponential cell growth (dashed black line, n = 5 colonies) and basal mean single cell fluorescence (green scatter plot, only every 6^th frame was measured, n = 5 colonies). In contrast, cells of the positive control experiment were initially exposed (> 1 sec) to maximal light intensity before starting time lapse imaging. Control cells (n = 271 cells) displayed immediate increase in the mean single cell fluorescence (pink scatter plot) and a stagnating total cell number (red dashed line) due to photo-oxidative stress. <b>(C)</b> Photobleaching was determined to be marginal as plotted as percentage of signal loss over mean single cell fluorescence (n = 85 cells).</p

Metabolic activity sensing of <i>C</i>. <i>glutamicum</i> wild type at intermittent nutrient limitation in minimal medium CGXII.

<p>Bacterial cells were cultivated in minimal medium CGXII with 4% glucose (CGXII + 4% GLC) at pH 7 before an indicated shift to a depletion phase by switch of perfusion medium supply. <b>(A)</b> Reference cultivation under continuous supply of CGXII + 4% GLG. <b>(B-D)</b> After 4 h pre-cultivation, the microchambers were perfused with minimal medium (n = 10 colonies). <b>(B)</b> without iron chelator protocatechuate (PCA) (CGXII + 4% GLC—PCA, marked with violet boxes, n = 10 colonies), <b>(C)</b> without iron (CGXII + 4% GLC—iron, marked with black boxes, n = 10 colonies), and <b>(D)</b> with carbon limitation (CGXII—PCA, marked with red boxes, n = 10 colonies), respectively. For carbon and iron depletion conditions the medium was switched back to initial medium after 15 h. Microcolony images of every cultivation conditions are shown at different experimental time points. The mean fluorescence of the colony and apparent growth rate over time are shown in comparison to the extracellular mean fluorescence of the perfusion medium in the supply channel (10 μm fluid height), inside the cultivation chamber entrances (1 μm fluid height) and in the direct cell proximity (1 μm fluid height).</p

Mean CAM conversion rate constant and mean calcein efflux rate constant.

<p><b>(A)</b> A mean maximal single cell reaction rate constant at 0.0025 min^-1 was determined for CAM conversion under inhibition of calcein efflux due to carbon limitation. A maximal mean single cell calcein efflux rate constant was determined to be approximately twice as high at 0.005 min^-1 (n = 5 colonies for each mean maximal single cell reaction rate constant). <b>(B)</b> Limitations in carbon supply inhibited cell division and energy driven transport of calcein out of the cell. Thus, mean single cell traces increase linearly over time until carbon supply was continued (every colored dotted line represent one individual cell, n = 15 cells).</p

Comparison of mean single cell fluorescence and apparent growth rate at different extracellular CAM concentrations.

<p>Mean calcein fluorescence for five extracellular CAM concentrations are shown for five cultivation chambers each (every chamber is indicated with a separate colour). Perfusion medium was switched from CGXII + 4% GLC to carbon free CGXII medium (CGXII—PCA) supply for 10 h (indicated with red frame) to inhibit the energy-dependent calcein efflux. CAM conversion by intracellular esterase activity and subsequent calcein fluorescence showed a non-linear fluorescence increase except for concentrations higher than the optimal extracellular CAM concentration of 46 μM. The corresponding apparent growth rates changed according to the carbon supply and not because of an increase CAM concentration.</p

Metabolic activity sensing under carbon limitation at single cell level.

<p><b>(A)</b> Microcolony image at the end of the cultivation phase under carbon limitation. Cells that performed no cell division after resupply of carbon are marked with arrows and are framed with dotted lines colored according to their mean single cell fluorescence traces in <b>(C). (B)</b> Microcolony image after regrowth under full nutrient supply at the end of cultivation. Cells that performed no cell division after carbon re-supply, are marked with arrows and are framed with dotted lines colored according to their mean single cell fluorescence traces in <b>(C). (C)</b> Mean single cell fluorescence traces of growing and non-growing bacteria before, during and after carbon depletion. Cells, which exhibit cell growth and division (grey lines), are compared to three phenotypes of non-growing cells with alternating mean single cell fluorescence during famine phase and reduced rate constants of calcein efflux under feast condition (red lines), increased mean single cell fluorescence during famine phase and after two hours after carbon resupply (blue lines) and average mean single cell fluorescence in comparison to normally dividing cells (black lines), respectively.</p

Metabolic activity sensing of growing and dividing cells.

<p><b>(A)</b> Non-growing cells can be classified into non-viable non-fluorescent cells and non-growing but metabolically active bacteria, which showed highest mean single cell fluorescence in comparison. Growing bacteria showed moderate to medium fluorescence. <b>(B-C)</b> Mean single cell fluorescence of freshly seeded and heterogeneous sized bacteria is given after a change to minimal medium CGXII + 4% GLC containing CAM (n = 6 cells). <b>(B)</b> Increase in the mean single cell fluorescence of six individual cells of different cell size is shown over time after infusion of CGXII + 4% GLC + 46 μM CAM. <b>(C)</b> The mean single cell fluorescence of the individual cells presented in (B) is given according their size during the short term experiment. Increase of cell size due to growth could was neglectable during the experimental time of 30 min. Mean single cell fluorescence in dependency of cell size revealed marginal differences of the final equilibrium mean fluorescence of individuals. <b>(D-F)</b> Metabolic activity sensing of cells grown in complex medium BHI at pH 6.6 (red), pH 7.0 (green), and pH 7.4 (blue), respectively. <b>(D)</b> Average values of 10 cultivation chambers are presented. No significant difference in calcein fluorescence (solid lines) and growth represented by total cell area (dashed lines) could be observed for cultivation of <i>C</i>. <i>glutamicum</i> at pH 7.0 and pH 7.4. At pH 6.6, however, increased mean calcein fluorescence as well as a reduced total cell area was observed (n = 10 colonies analyzed at each pH). <b>(E)</b> The mean single cell area indicated a tendency of cell size reduction in average over time (n = 10 colonies analyzed at each pH). <b>(F)</b> Mean fluorescence correlates positively with the mean single cell area at all three pH values (n = 10 colonies analyzed at each pH).</p

Microfluidic device and its submicrostructures.

<p><b>(A)</b> Assembled microfluidic device with connected inlet and outlet tubings. <b>(B)</b> Parallel cultivation chamber arrays with branched main channels that subdivide in smaller channels for media perfusion. A a single device contains four cultivation chamber arrays with 352 chambers each. <b>(C)</b> Microcultivation chamber. <b>(D)</b> SEM micrograph of a cultivation chamber.</p

Cellular CAM metabolism model of a <i>C</i>. <i>glutamicum</i> cell.

<p>Gram positive <i>C</i>. <i>glutamicum</i> has a cell wall of different layers, which has to be passed by the fluorogenic substrate CAM. Once the cell wall is transversed by a presumably non-passive diffusive mechanism, CAM is converted to the fluorophore calcein and ethanol by intracellular carboxylesterases. The acidic calcein is assumed to be secreted by an energy dependent transport mechanism with a putative ATPase.</p