细胞内pH和极性监测对于理解细胞过程和功能至关重要。这项研究采用pH和极性敏感的纳米材料,如碳点(CD)用于细胞内pH值的传感,极性,和粘度使用积分时间分辨荧光各向异性(FA)成像(TR-FAIM)和荧光寿命(FLT)成像显微镜(FLIM),从而实现全面表征。CD表面的官能团对微环境的变化表现出敏感性,导致荧光强度(FI)和FLT根据pH和极性的变化。在2-8的pH范围内,水溶液中CD的FLT从6.38±0.05ns逐渐变为8.03±0.21ns。有趣的是,在测量极性降低的CD时,观察到FI和FLT的复杂关系。然而,FA和旋转相关时间(θ)从0.062±0.019增加到0.112±0.023,从0.49±0.03ns增加到2.01±0.27ns,分别。FA和θ的增加归因于伴随极性降低的较高粘度。此外,发现CD结合到大肠杆菌的三个位置:细胞壁,内膜,和细胞质,使用FI和FA衰变成像实现细胞内表征。FLT提供了细胞质pH值(7.67±0.48)的见解,同意以前的作品,以及细胞壁和内膜极性的降低。根据FA,某些地区怀疑CD聚集,和θ提供了有关由于与生物分子的聚集和/或相互作用而导致的细胞质异质性的信息。组合的TR-FAIM/FLIM系统允许同时监测通过FLIM的pH和极性变化以及通过TR-FAIM的粘度变化。
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.