Bodipy-FL-ATP (10 μM) diffusion into a cardiac myocyte from a patch pipette (on-cell pipette resistance, 3 MΩ 24°C). (A–C) Fluorescence micrographs of the myocyte and patch pipette tip just before opening the myocyte (A), after 3 min (B), and after 40 min (C). Dashed lines indicate the positions of the fluorescence intensity profiles displayed in E and F. Intensities were normalized to a maximal cytoplasmic value in each experiment as arbitrary fluorescence units (AFU). (D) The time course of total cytoplasmic fluorescence (red), fitted to an exponential function (gray; time constant, 17 min), and the time course of cytoplasmic fluorescence immediately in front of the pipette tip (blue, F₀). The average time constant for four experiments was 42 min. (E) Longitudinal fluorescence line scans along the myocyte at 0, 3, 15, and 40 min. Note that a longitudinal gradient is still evident after 40 min. F₀ indicates the position of data points in D. (F) Line scan through the pipette tip and across the width of the myocyte, as indicated in A and C, at 0 and 40 min. As described in Materials and methods, a fluorescence peak (gray) often formed in the pipette tip over 40 min. This corresponds to a fluorophore condensate, which had in our experience no evident effect on experimental results. Fluorescence of the myocyte cytoplasm remained <50% of the pipette tip fluorescence, similar to three equivalent experiments. (G and H) Simulation of Bodipy-FL-ATP diffusion into a myocyte assumed to be 120 μm long. From the four myocytes studied, equilibration was the fastest in this myocyte. Therefore, the simulated diffusion coefficient obtained, 6 × 10⁻⁸ cm²/s, is the maximal coefficient justified by experimental results. (G) Simulated mean cytoplasmic fluorophore concentration (red) and fluorophore concentration in the first compartment at the pipette tip ([X]₀). Accurate simulation was obtained with the time constant of dye diffusion through the entire cytoplasm, approximated as $λmyo2/2 × Dx$⁠, being approximately threefold greater than the time constant of dye exchange between the pipette tip and the cytoplasm, 1/K_pip (Eq. 3). This reflects reasonably the ratio of time constants for F₀ and the mean myocyte fluorescence in D. (H) Simulated cytoplasmic concentration profiles at the given time points after initiating diffusion from the pipette tip.