Differentiation-defined cellular states reveal differences not captured by mean JC-1 fluorescence ratios

JC-1 fluorescence is widely used to assess mitochondrial membrane potential (ΔΨm) and is typically summarized as a mean red/green ratio, which may not fully capture spatial variability within images. However, ΔΨm is spatially heterogeneous at the subcellular level, raising the possibility that mean-based analysis alone may overlook differences between cellular states. JC-1 imaging was performed in undifferentiated and differentiated SH-SY5Y cells. Mean JC-1 red/green fluorescence ratios and the coefficient of variation (CV) were calculated from identical image fields. Differentiation induced a neuronal-like state characterized by tyrosine hydroxylase (TH) positivity (95.33% ± 0.64% vs. 0%). Mean JC-1 red/green fluorescence ratios showed no significant difference between groups (4.77 ± 1.33 vs. 4.92 ± 1.17; p = 0.806). In contrast, the CV decreased significantly after differentiation (58.49% ± 4.42% vs. 48.77% ± 3.84%; p < 0.001), indicating reduced signal dispersion. These findings indicate that mean JC-1 ratios alone may fail to detect differences between cellular states. Dispersion analysis provides complementary information and may be particularly useful when fluorescence heterogeneity differs between cellular states.