Fluorescence lifetime imaging microenvironmental dynamics in mitochondria and nucleolus during stimuli stress in living cells

Abstract

Nucleolus and mitochondria play an important role in the maintenance of cell balance, and studying their physiological processes is helpful to understand the biological functions. In this paper, a red fluorescent pyrene rhodamine probe was used to target and label cell mitochondria and nucleolus under different conditions, and the binding mode of probe and RNA was also clarified by bio-computational simulation results. Confocal laser scanning microscopy was used to analyze the morphological changes of apoptosis in HeLa cells under the action of laser light, paclitaxel and colchicine, and the changes of micro-environmental viscosity between mitochondria and nucleolus were quantitatively analyzed by fluorescence lifetime imaging phase map. It was determined that the average fluorescence lifetime of mitochondria labeled by probes in steady-state HeLa cells was about 3.65 ns. The mitochondrial viscosity is about 66 cP. After laser irradiation, mitochondrial fracture and fusion occurred, the fluorescence lifetime of the probe increased to 3.82 ns and the mitochondrial viscosity increased to about 131 cP. The mean fluorescence lifetime of the nucleolus of HeLa cells was increased from 4.23 ns to 4.32 ns, indicating the changes of the nucleolus and mitochondrial microenvironment induced by prolonged laser irradiation. Apoptosis was induced by paclitaxel and colchicine, and the nucleolus moved out of the nucleus and into the cytoplasm. Meanwhile, the fluorescence lifetime of nucleolus and mitochondria labeled by the probe increased first and then decreased. The time of paclitaxel treatment increased from 0.5 h to 4 h, and the average lifetime of nucleolus of HeLa cells labeled by the probe increased from 4.19 ns to 4.47 ns, and finally decreased to 4.42 ns, reflecting the difference of nucleolar of HeLa cells microenvironment induced by apoptosis induced by different treatment time of paclitaxel. Compared with the blank HeLa cell, the average lifetime of the probe increased from 4.10 ns to 4.34 ns after 1 h treatment with colchicine at low concentration (10 nM), and continuously increased to 4.47 ns after 1 h treatment with high concentration (100 nM) colchicine. The microenvironment of nucleolus and mitochondria induced by apoptosis induced by colchicine at different concentrations was reflected. The above three ways of inducing apoptosis by laser light, paclitaxel and colchicine proved that the changes of nucleolar and mitochondrial microenvironment and functional changes of HeLa cells under the condition of cell instability provided a new research method for the study of the dynamic process of apoptosis induced by different pathways and the study of diseases related to nucleolar and mitochondrial dysfunction.