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).