Estimation of the Mitochondrial Membrane Potential Using Fluorescence Lifetime Imaging Microscopy.

Area of Science:

• Cellular biology
• Microscopy
• Biophysics

Background:

• Cell metabolism monitoring is crucial for understanding cellular function.
• Fluorescence lifetime imaging microscopy (FLIM) offers advanced capabilities for cellular analysis.
• Assessing mitochondrial membrane potential (MMP) is key to evaluating mitochondrial function.

Purpose of the Study:

• To evaluate SYTO and tetramethylrhodamine methyl ester (TMRM) as fluorescent probes for MMP assessment using FLIM.
• To demonstrate the utility of these dyes in complex cellular models, including 3D cultures and in vivo applications.
• To enable improved cell type discrimination and functional analysis based on mitochondrial activity.

Main Methods:

• Utilized fluorescence lifetime imaging microscopy (FLIM) for cellular analysis.
• Employed SYTO (green/orange-emitting) and tetramethylrhodamine methyl ester (TMRM) fluorescent dyes.
• Applied the dyes to human colon cancer HCT116 cells and Lgr5-GFP mouse intestinal organoids (3D cultures).
• Performed multiplexed imaging combining Lgr5-GFP, Hoechst 33342, and TMRM-FLIM.

Main Results:

• SYTO dyes exhibited both nuclear and mitochondrial accumulation.
• FLIM analysis revealed heterogeneity in mitochondrial polarization during the cell cycle of HCT116 cells.
• SYTO and TMRM dyes demonstrated efficient staining and compatibility with two-photon excitation in 3D organoid cultures.
• Multiplexed imaging successfully identified metabolically active cells within the stem cell niche.
• TMRM-FLIM visualized distinct membrane potential differences between Lgr5-positive stem cells and other cell types.

Conclusions:

• SYTO 24 and TMRM are promising fluorescent markers for advanced FLIM-based studies of cell bioenergetics.
• These dyes facilitate the study of mitochondrial function in complex 3D and in vivo models.
• The findings support the use of these probes for improved cell segmentation and functional discrimination.