2. Academic Validation
  3. Imaging mitochondrial membrane potential via concentration-dependent fluorescence lifetime changes

Imaging mitochondrial membrane potential via concentration-dependent fluorescence lifetime changes

  • Nat Commun. 2025 Dec 12;16(1):11088. doi: 10.1038/s41467-025-66042-x.
Dilizhatai Saimi 1,  Luc Reymond 2,  Tursunjan Aziz 3,  Xuan Shen 4,  Ziying Luo 1 5,  Shuaibo Pi 1,  Yitong Liu 1 5,  Song Fu 6 7 8,  Shuangjin Ding 1,  Anming Meng 3 9 10,  Liangyi Chen 1 11 12,  Hui Jiang 7 8 13,  Zhixing Chen 14 15 16 17 18
Affiliations

Affiliations

  • 1 College of Future Technology, Institute of Molecular Medicine, National Biomedical Imaging Center, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Peking University, Beijing, China.
  • 2 Biomolecular Screening Facility, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.
  • 3 Laboratory of Molecular Developmental Biology, State Key Laboratory of Membrane Biology, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China.
  • 4 College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing, China.
  • 5 Peking-Tsinghua Center for Life Science, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China.
  • 6 Graduate School of Peking Union Medical College, Beijing, China.
  • 7 National Institute of Biological Sciences, Beijing, China.
  • 8 Beijing Key Laboratory of Cell Biology for Animal Aging, Beijin, China.
  • 9 Developmental Diseases and Cancer Research Center, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China.
  • 10 Laboratory of Stem Cell Regulation, Guangzhou Laboratory, Guangzhou, China.
  • 11 PKU-Nanjing Institute of Translational Medicine, Nanjing, China.
  • 12 State Key Laboratory of Membrane Biology, Peking University, Beijing, China.
  • 13 Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing, China.
  • 14 College of Future Technology, Institute of Molecular Medicine, National Biomedical Imaging Center, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Peking University, Beijing, China. [email protected].
  • 15 Peking-Tsinghua Center for Life Science, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China. [email protected].
  • 16 PKU-Nanjing Institute of Translational Medicine, Nanjing, China. [email protected].
  • 17 State Key Laboratory of Membrane Biology, Peking University, Beijing, China. [email protected].
  • 18 GenVivo Tech, Nanjing, China. [email protected].
PMID: 41387403 DOI: 10.1038/s41467-025-66042-x
Abstract

Mitochondria are central to cellular metabolism. Various fluorescence tools have been developed for imaging the mitochondrial environment. Yet, new reporters and imaging methods for directly reading the mitochondrial status are needed for high spatial-temporal resolution imaging. Here, we introduce PK Mito Deep Red (PKMDR), a low-phototoxicity mitochondrial probe for time-lapse imaging, whose fluorescence lifetime serves as a sensitive indicator of mitochondrial membrane potential (Δψm). The positively charged PKMDR accumulates within mitochondria under a higher Δψm, leading to concentration-induced quenching and a measurable decrease in fluorescence lifetime. Since mitochondrial respiration primarily regulates Δψm, PKMDR's fluorescence lifetime effectively reports on the status of Oxidative Phosphorylation. Using PKMDR with fluorescence lifetime imaging microscopy (FLIM), we visualize heterogeneous Δψm across individual cells, organoids, and tissues over time. This method reliably reveals the heterogeneity between metabolically active peripheral mitochondria and relatively inactive perinuclear mitochondria in various cell types. Overall, PKMDR-FLIM is a robust tool for directly visualizing Δψm with high spatiotemporal resolution.

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