Huang Maojia, Liang Xinyue, Zhang Zixiao, Wang Jing, Fei Yiyan, Ma Jiong, Qu Songnan, Mi Lan
Department of Optical Science and Engineering, Shanghai Engineering Research Center of Ultra-precision Optical Manufacturing, Green Photoelectron Platform, Fudan University, Shanghai 200433, China.
State Key Laboratory of High Field Laser Physics, Shanghai Institute of Optics and Fine Mechanics, Chinese Academy of Sciences, Shanghai 201800, China.
Nanomaterials (Basel). 2020 Mar 25;10(4):604. doi: 10.3390/nano10040604.
The monitoring of intracellular pH is of great importance for understanding intracellular trafficking and functions. It has various limitations for biosensing based on the fluorescence intensity or spectra study. In this research, pH-sensitive carbon dots (CDs) were employed for intracellular pH sensing with fluorescence lifetime imaging microscopy (FLIM) for the first time. FLIM is a highly sensitive method that is used to detect a microenvironment and it can overcome the limitations of biosensing methods based on fluorescence intensity. The different groups on the CDs surfaces changing with pH environments led to different fluorescence lifetime values. The CDs aqueous solution had a gradual change from 1.6 ns to 3.7 ns in the fluorescence lifetime with a pH range of 2.6-8.6. Similar fluorescence lifetime changes were found in pH buffer-treated living cells. The detection of lysosomes, cytoplasm, and nuclei in living cells was achieved by measuring the fluorescence lifetime of CDs. In particular, a phasor FLIM analysis was used to improve the pH imaging. Moreover, the effects of the coenzymes, amino acids, and proteins on the fluorescence lifetime of CDs were examined in order to mimic the complex microenvironment inside the cells.
监测细胞内pH值对于理解细胞内运输和功能非常重要。基于荧光强度或光谱研究的生物传感存在各种局限性。在本研究中，首次将pH敏感碳点（CDs）用于通过荧光寿命成像显微镜（FLIM）进行细胞内pH传感。FLIM是一种用于检测微环境的高灵敏度方法，它可以克服基于荧光强度的生物传感方法的局限性。CDs表面的不同基团随pH环境变化导致不同的荧光寿命值。CDs水溶液在pH范围为2.6 - 8.6时，荧光寿命从1.6 ns逐渐变化到3.7 ns。在pH缓冲处理的活细胞中也发现了类似的荧光寿命变化。通过测量CDs的荧光寿命实现了对活细胞中溶酶体、细胞质和细胞核的检测。特别是，采用相量FLIM分析来改善pH成像。此外，还研究了辅酶、氨基酸和蛋白质对CDs荧光寿命的影响，以模拟细胞内复杂的微环境。
