Figure S3.
Baseline-normalized ratiometry rules out motion and focus artifacts, confirming that beat-locked mito-iATP oscillations are genuine metabolic signals. (A and C) Representative recordings from intact SA node preparations exhibiting Mode 1 (A) and Mode 2 (C) mito-iATP dynamics. The ATP sensor channel (mito-iATP, green) is shown together with a spectrally distinct reference channel (JFX554–HaloTag, red); the baseline-normalized ratiometric trace (R/R0, orange) is overlaid. Traces are shown as normalized fluorescence (F/F0; baseline = 1.0; dashed line). Data in A–D were obtained from three independent SA node preparations (N = 3); 26 events are shown in B and 22 events in D. (B and D) Event-level relationship between mito-iATP peak amplitude (ΔF/F0) and ratiometric peak amplitude (ΔR/R0) for Mode 1 (B) and Mode 2 (D) events. Each symbol represents one mito-iATP transient event. Solid lines indicate linear fits to Mode 1 (slope = 1.07, R2 = 0.92) and Mode 2 (slope = 1.09, R2 = 0.97) events, with shaded regions denoting 95% confidence intervals. Signals were extracted from fixed ROIs and analyzed as F/F0 (baseline = 1.0). The ratiometric signal was computed as (R/R0 = (Fmito-iATP/Fmito-iATP,0)/(FHaloTag/FHaloTag,0)). (E) Representative recording during controlled axial displacement (Δz-step = 5 µm) in an intact SA node preparation. The shaded region indicates the imposed z-step interval. Mito-iATP (green) and JFX554–HaloTag (red) are shown as F/F0 (baseline = 1.0), with (R/R0) overlaid (orange). (F) Within this representative node, peak absolute deviation from baseline during the z-step interval is summarized for mito-iATP (|ΔF/F0|), JFX554–HaloTag (|ΔF/F0|), and the ratiometric signal (|Δ(R/R0)|). Each symbol represents one ROI/cell measured within the same preparation (N = 1 SA node; n = 9 ROIs/cells). N represents the number of independent mice. Refer to the image caption for details. Panel A: Representative line traces compare the kinetics of whole-node mito-iATP transients. The graph includes three lines: a green line representing mito-iATP Mode 1, a red line representing JFX554–HaloTag ligand, and an orange line representing the ratio of mito-iATP Mode 1 to JFX554–HaloTag ligand. The x-axis represents time in milliseconds (m s), and the y-axis represents normalized fluorescence and normalized ratio. Panel B: A scatter plot shows the relationship between mito-iATP peak amplitude and ratiometric peak amplitude for Mode 1 events. Each symbol represents one mito-iATP transient event, with a solid line indicating a linear fit and shaded regions denoting 95 percent confidence intervals. The x-axis represents mito-iATP change in fluorescence relative to baseline, and the y-axis represents change in ratio relative to baseline. Panel C: Representative line traces compare the kinetics of whole-node mito-iATP transients for Mode 2 events. The graph includes three lines: a green line representing mito-iATP Mode 2, a red line representing JFX554–HaloTag ligand, and an orange line representing the ratio of mito-iATP Mode 2 to JFX554–HaloTag ligand. The x-axis represents time in milliseconds (ms), and the y-axis represents normalized fluorescence and normalized ratio. Panel D: A scatter plot shows the relationship between mito-iATP peak amplitude and ratiometric peak amplitude for Mode 2 events. Each symbol represents one mito-iATP transient event, with a solid line indicating a linear fit and shaded regions denoting 95 percent confidence intervals. The x-axis represents mito-iATP change in fluorescence relative to baseline, and the y-axis represents change in ratio relative to baseline. Panel E: Representative line traces show a representative recording during controlled axial displacement (z-step) in an intact SA node preparation. The graph includes three lines: a green line representing mito-iATP, a red line representing JFX554–HaloTag ligand, and an orange line representing the ratio of mito-iATP to JFX554–HaloTag ligand. The x-axis represents time in milliseconds, and the y-axis represents normalized fluorescence (F/F0) and normalized ratio. The shaded region indicates the imposed z-step interval. Panel F: A scatter plot summarizes the peak absolute deviation from baseline during the z-step interval for mito-iATP, JFX554–HaloTag ligand, and the ratiometric signal. Each symbol represents one region of interest (ROI) or cell measured within the same preparation.

Baseline-normalized ratiometry rules out motion and focus artifacts, confirming that beat-locked mito-iATP oscillations are genuine metabolic signals. (A and C) Representative recordings from intact SA node preparations exhibiting Mode 1 (A) and Mode 2 (C) mito-iATP dynamics. The ATP sensor channel (mito-iATP, green) is shown together with a spectrally distinct reference channel (JFX554–HaloTag, red); the baseline-normalized ratiometric trace (R/R0, orange) is overlaid. Traces are shown as normalized fluorescence (F/F0; baseline = 1.0; dashed line). Data in A–D were obtained from three independent SA node preparations (N = 3); 26 events are shown in B and 22 events in D. (B and D) Event-level relationship between mito-iATP peak amplitude (ΔF/F0) and ratiometric peak amplitude (ΔR/R0) for Mode 1 (B) and Mode 2 (D) events. Each symbol represents one mito-iATP transient event. Solid lines indicate linear fits to Mode 1 (slope = 1.07, R2 = 0.92) and Mode 2 (slope = 1.09, R2 = 0.97) events, with shaded regions denoting 95% confidence intervals. Signals were extracted from fixed ROIs and analyzed as F/F0 (baseline = 1.0). The ratiometric signal was computed as (R/R0 = (Fmito-iATP/Fmito-iATP,0)/(FHaloTag/FHaloTag,0)). (E) Representative recording during controlled axial displacement (Δz-step = 5 µm) in an intact SA node preparation. The shaded region indicates the imposed z-step interval. Mito-iATP (green) and JFX554–HaloTag (red) are shown as F/F0 (baseline = 1.0), with (R/R0) overlaid (orange). (F) Within this representative node, peak absolute deviation from baseline during the z-step interval is summarized for mito-iATP (|ΔF/F0|), JFX554–HaloTag (|ΔF/F0|), and the ratiometric signal (|Δ(R/R0)|). Each symbol represents one ROI/cell measured within the same preparation (N = 1 SA node; n = 9 ROIs/cells). N represents the number of independent mice.

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