Nucleolus and mitochondria play an important role in maintaining cell balance, and studying their physiological processes is helpful in understanding the biological functions. In this work, a red fluorescent pyrene rhodamine probe is used to target and label cell mitochondria and nucleolus under different conditions, and the binding mode of probe and RNA is also clarified by bio-computational simulation results. Confocal laser scanning microscopy is used to analyze the morphological changes of apoptosis in HeLa cells under the action of laser light, paclitaxel and colchicine, and the changes of microenvironment between mitochondria and nucleolus are quantitatively analyzed by fluorescence lifetime imaging phase map. It is determined that the average fluorescence lifetime of the probe labeled mitochondria in steady-state HeLa cells is about 3.65 ns. The mitochondrial viscosity is about 66×10^–3 Pa·s. After laser irradiation, mitochondrial fracture and fusion occur, the fluorescence lifetime of the probe decreases to 3.61 ns and the mitochondrial viscosity increases to about 131×10^–3 Pa·s. The mean fluorescence lifetime of the probe labeled nucleolus of HeLa cells increases from 4.23 ns to 4.32 ns, indicating that the changes of the nucleolus microenvironment is induced by prolonging laser irradiation. Apoptosis is induced by paclitaxel and colchicine, and the nucleolus moves out of the nucleus and into the cytoplasm. Meanwhile, the fluorescence lifetime of the probe labeled nucleolus first increases and then decreases. The treatment time of paclitaxel increases from 0.5 h to 4 h, and the average lifetime of the probe labeled nucleolus of HeLa cells increases from 4.19 ns to 4.47 ns, and finally decreases to 4.42 ns, reflecting the differences in nucleolar microenvironment of HeLa cells induced by different treatment times of paclitaxel. Comparing with the blank HeLa cell, the average lifetime of the probe increases from 4.10 ns to 4.34 ns after 1 h treatment with colchicine at low concentration (10 nmol/L), and continuously increases to 4.47 ns after 1 h treatment with high concentration (100 nmol/L) colchicine. The microenvironments of nucleolus and mitochondria induced by apoptosis induced by colchicine at different concentrations are shown. The above three ways of inducing injury or apoptosis, i.e. by laser light, paclitaxel and colchicine, prove that the changes of nucleolar and mitochondrial microenvironment and functional changes of HeLa cells under the condition of cell instability provide a new method of studying the dynamic process of apoptosis induced by different pathways and the diseases related to nucleolar and mitochondrial dysfunction as well.