Probing Polarity and pH Sensitivity of Carbon Dots in Escherichia coli through Time-Resolved Fluorescence Analyses

Intracellular monitoring of pH and polarity is crucial for understanding cellular processes and functions. This study employed pH- and polarity-sensitive nanomaterials such as carbon dots (CDs) for the intracellular sensing of pH, polarity, and viscosity using integrated time-resolved fluorescence anisotropy (FA) imaging (TR-FAIM) and fluorescence lifetime (FLT) imaging microscopy (FLIM), thereby enabling comprehensive characterization. The functional groups on the surface of CDs exhibit sensitivity to changes in the microenvironment, leading to variations in fluorescence intensity (FI) and FLT according to pH and polarity. The FLT of CDs in aqueous solution changed gradually from 6.38 ± 0.05 ns to 8.03 ± 0.21 ns within a pH range of 2-8. Interestingly, a complex relationship of FI and FLT was observed during measurements of CDs with decreasing polarity. However, the FA and rotational correlation time (θ) increased from 0.062 ± 0.019 to 0.112 ± 0.023 and from 0.49 ± 0.03 ns to 2.01 ± 0.27 ns, respectively. This increase in FA and θ was attributed to the higher viscosity accompanying the decrease in polarity. Furthermore, CDs were found to bind to three locations in Escherichia coli: the cell wall, inner membrane, and cytoplasm, enabling intracellular characterization using FI and FA decay imaging. FLT provided insights into cytoplasmic pH (7.67 ± 0.48), which agreed with previous works, as well as the decrease in polarity in the cell wall and inner membrane. The CD aggregation was suspected in certain areas based on FA, and the θ provided information on cytoplasmic heterogeneity due to the aggregation and/or interactions with biomolecules. The combined TR-FAIM/FLIM system allowed for simultaneous monitoring of pH and polarity changes through FLIM and viscosity variations through TR-FAIM.