Figure S6.
Ratiometric validation of mito-iATP signals in isolated SA node myocytes rules out optical artifacts, confirming that Mode 1 and Mode 2 transients reflect genuine metabolic signals. (A) Representative fluorescence image of an isolated SA node myocyte expressing mito-iATP (green) and labeled with the reference fluorophore JFX554-HaloTag ligand (red). Scale bar, 5 µm. (B and D) Representative single-cell recordings showing Mode 1 mito-iATP transients (B) and Mode 2 mito-iATP transients (D). The baseline-normalized mito-iATP signal (F/F0; green) is shown alongside the reference fluorophore signal (JFX554-HaloTag; red) and the resulting ratiometric trace (R/R0; orange). Dashed lines denote baseline fluorescence. Scale bars: 200 ms. (C and E) Event-level relationship between the mito-iATP peak amplitude (F/F0) and the ratiometric amplitude (R/R0) for Mode 1 (C) and Mode 2 (E) events. Each blue circle represents an individual transient. Solid lines indicate linear fits (Mode 1: slope = 0.98, R2 = 0.95; Mode 2: slope = 0.99, R2 = 0.98), with shaded regions representing 95% confidence intervals. Data in A–E were obtained from SA node cells isolated from three mice (N = 3); C shows 21 events, and E shows 27 events. N represents the number of independent mice. Refer to the image caption for details. Panel A: Fluorescence image showing mito-iATP and reference fluorophore distribution. Panel B: Representative confocal line scan showing Mode 1 mito-iATP transient. The horizontal axis represents time in milliseconds, and the vertical axis represents normalized fluorescence. Panel C: Scatter plot showing correlation between mito-iATP and ratiometric amplitudes. Each blue circle represents an individual transient. The solid line indicates a linear fit with a slope of 0.98 and R-squared value of 0.95. Panel D: Representative confocal line scan showing Mode 2 mito-iATP transient. The horizontal axis represents time in milliseconds, and the vertical axis represents normalized fluorescence. Panel E: Scatter plot showing strong correlation for Mode 2 amplitudes. Each blue circle represents an individual transient. The solid line indicates a linear fit with a slope of 0.99 and an R-squared value of 0.98.

Ratiometric validation of mito-iATP signals in isolated SA node myocytes rules out optical artifacts, confirming that Mode 1 and Mode 2 transients reflect genuine metabolic signals. (A) Representative fluorescence image of an isolated SA node myocyte expressing mito-iATP (green) and labeled with the reference fluorophore JFX554-HaloTag ligand (red). Scale bar, 5 µm. (B and D) Representative single-cell recordings showing Mode 1 mito-iATP transients (B) and Mode 2 mito-iATP transients (D). The baseline-normalized mito-iATP signal (F/F0; green) is shown alongside the reference fluorophore signal (JFX554-HaloTag; red) and the resulting ratiometric trace (R/R0; orange). Dashed lines denote baseline fluorescence. Scale bars: 200 ms. (C and E) Event-level relationship between the mito-iATP peak amplitude (F/F0) and the ratiometric amplitude (R/R0) for Mode 1 (C) and Mode 2 (E) events. Each blue circle represents an individual transient. Solid lines indicate linear fits (Mode 1: slope = 0.98, R2 = 0.95; Mode 2: slope = 0.99, R2 = 0.98), with shaded regions representing 95% confidence intervals. Data in A–E were obtained from SA node cells isolated from three mice (N = 3); C shows 21 events, and E shows 27 events. N represents the number of independent mice.

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