Imaging mitochondrial membrane potential via concentration-dependent fluorescence lifetime changes

Area of Science:

• Cell Biology
• Biochemistry
• Biophysics

Background:

• Mitochondria are vital for cellular metabolism and energy production.
• Existing fluorescence tools for mitochondrial imaging have limitations in spatial-temporal resolution.
• Direct reporters for mitochondrial status, particularly membrane potential, are needed.

Purpose of the Study:

• To introduce PK Mito Deep Red (PKMDR), a novel fluorescent probe for mitochondrial imaging.
• To establish PKMDR's fluorescence lifetime as a sensitive indicator of mitochondrial membrane potential (Δψm).
• To demonstrate the utility of PKMDR-based fluorescence lifetime imaging microscopy (FLIM) for visualizing metabolic heterogeneity.

Main Methods:

• Development and characterization of PKMDR, a low-phototoxicity mitochondrial probe.
• Utilizing fluorescence lifetime imaging microscopy (FLIM) to measure Δψm.
• Application of PKMDR-FLIM in live cells, organoids, and tissues for time-lapse imaging.

Main Results:

• PKMDR fluorescence lifetime is a sensitive indicator of Δψm, correlating with mitochondrial respiration and oxidative phosphorylation.
• PKMDR-FLIM effectively visualizes heterogeneous Δψm across individual cells, organoids, and tissues.
• The method reveals metabolic differences between peripheral and perinuclear mitochondria.

Conclusions:

• PKMDR is a robust fluorescent probe for assessing mitochondrial membrane potential.
• PKMDR-FLIM provides high spatiotemporal resolution for visualizing mitochondrial metabolic status.
• This technique offers new insights into cellular and tissue metabolic heterogeneity.