Fluorescence-lifetime optical electrophysiology in contracting cardiomyocytes

Figures

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  Fig. 1.. Overview of cardiac electrophysiology and fluorescence lifetime imaging system. (A) Diagram showing the excitation–contraction coupling mechanism in cardiac cells. Adapted from ref. 2. (B) Schematic of fluorescence lifetime imaging microscopy experimental setup. (C) Timing diagram showing the gated operation of the SPAD camera. The antigate profile is obtained by subtracting the gated images from the intensity frames. (D) Magnified view showing the gated capture of a single fluorescence emission. Shaded regions under the green curve highlight the regions integrated by each SPAD channel. (E) Multiple binary (1-bit) SPAD frames are accumulated and summed to form an 8-bit intensity image. Each binary exposure of 10.24 μs represents the total fluorescence signal integrated across 819 laser excitations. (F) Example 8-bit fluorescence intensity image of a hiPSC-CM monolayer captured by the SPAD. (Scale bar, 100 μm.) (G) Immunofluorescence image of hiPSC-CM monolayer stained for Wheat-Germ Agglutinin-Alexa Fluor 488 (membranes) and DAPI (nuclei). White (Scale bar, 50 μm.) Insets show magnified views of two regions indicated in the main panel. (Scale bar, 15 μm.)

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  Fig. 2.. Comparison of fluorescence lifetime and intensity voltage measurements highlights motion-robust signals from lifetime imaging in human cardiac monolayers. (A) Excitation–contraction time traces in a hiPSC-CM monolayer paced at 1 Hz under natural contractile conditions (green) and after treatment with BDM (pink). Top to bottom: contraction waveforms, APs measured with intensity, and APs measured with fluorescence lifetime. Traces are normalized for comparison; contraction traces are normalized to the pretreatment state to highlight changes in contraction amplitude. (B–E) Normalized, time-averaged AP waveforms from intensity- (B and D) and lifetime-based (C and E) measurements. Individual traces are overlaid in a lighter shade to illustrate variability. (F) Box plot analysis of the reduction in contraction amplitude induced by BDM treatment. (G and H) Box plot showing the change in APD30/APD90 before and after BDM treatment measured with fluorescence intensity (G) and fluorescence lifetime (H). For (E–G) the boxes show the IQR, the horizontal lines represent medians, and the whiskers extend to 1.5× the IQR. For all statistical tests, two-sample independent t tests were analyzed…

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  Fig. 3.. Calibrated mapping of voltage dynamics in intact human cardiac monolayers. (A) Spatially resolved map of the estimated RMP during diastole. (B) Corresponding spatially averaged AP time trace from the FOV shown in (A). (C) Temporally averaged and normalized waveforms from (B) recorded with fluorescence lifetime and intensity. (D–I) Equivalent analyses for 3 additional regions from two distinct monolayers: (D and F) correspond to one monolayer, while (A and H) correspond to another. (J) Two-step masking strategy used to identify regions with low signal or nonphysiological RMP values. An intensity-based mask excludes low-signal regions, and an RMP-based mask highlights regions with diastolic values between −60 and −100 mV. (K) The combined mask illustrates regions consistent with expected physiological ranges. (L–O) Example masked maps shown for visualization purposes. All analyses in (A–I) are performed on unmasked, spatially averaged data. (Scale bar, 50 μm.)

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  Fig. 4.. Distributions of diastolic and peak membrane potential across FOVs. (A–D) Histograms of diastolic membrane potential (RMP) measured across regions corresponding to the FOVs shown in Fig. 3 A, D, F, and H, respectively. Diastolic values were computed by averaging Vm over multiple frames during resting periods for each FOV. Also shown are the corresponding histograms of peak Vm values, extracted by identifying the peak frame of each AP firing event within each FOV and averaging across multiple events. Insets show the corresponding FOVs. (Scale bar, 50 μm.)

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  Fig. 5.. Spatiotemporal mapping of calcium wave propagation in hiPSC-CM monolayers by widefield fluorescence lifetime imaging. (A and B) Representative fluorescence intensity (A) and lifetime (B) images of a hiPSC-CM monolayer over a 1.6×1.6-mm FOV. (Scale bar, 400 μm.) (C and D) Polar plots showing percentage deviation from mean intensity (C) and lifetime (D) across the images in (A and B) respectively. (E) Plot of lifetime versus intensity. The red shaded region highlights the low-intensity regime, where lifetime estimates show greater variability due to lower photon counts. (F) Representative calcium transient time trace with propagated uncertainty in inferred Ca2+ concentration (gray shading), derived from the calibration lifetime measurement error. (G) Activation map of the first beat highlighted in pink in (F) showing the time of calcium activation at each spatial region (Tact.). The activation wave propagates from top to bottom. (H) ΔF/F0 traces from two regions of interest highlighted in (G), and (I) the corresponding lifetime and estimated Ca2+ concentration traces. (J–L) Same as (G–I) for the second beat highlighted in green in (F). The activation wave now propagates from…