Figure 6. Cardiac sarcomere imaging with image sequence reconstruction. (A) Data showing the method of image sequence reconstruction for in vivo sarcomere imaging. Shown in the following order from top to bottom (analyzed by PC1; see Fig. 1 A): ECG, LVP, objective lens position (along the Z-axis), and EMCCD camera shutter signal (either “open” or “closed” at 100 Hz). Data obtained 5, 20, and 42 s (from left to right) after the onset of recording (0.5 s at each time point). The objective lens position was moved downward by a piezo actuator at a 1-µm increment along the Z-axis; i.e., 0, 3, and 7 µm at 5-, 20-, and 42-s time points, respectively, after the onset of recording (see B for captured static images). HR, ∼400 bpm in this animal throughout the observation. (B; top) Time course of a change in LVP during the cardiac cycle (divided into 17 phases, from −8 to 8, with a period of one phase at 10.2 ms). (Middle) Images of an AcGFP-expressing myocyte in the LV in vivo captured at phases −8, −2, 0, and 7 at 0, 2, and 6 µm in the Z-direction. (Bottom) Best-focused images shown by asterisks in middle. SL was analyzed by using the multiple Gaussian fitting (see Fig. S4, A–D). SL values were 1.75 ± 0.11 (74), 1.64 ± 0.11 (68), 1.63 ± 0.10 (68), and 1.74 ± 0.10 µm (82) at phases −8, −2, 0, and 7, respectively (number of sarcomeres shown in parentheses). Bar, 20 µm. (C) Time-dependent changes in SL (red) and LVP (black) obtained from the analysis in B using the reconstructed image sequence. SL values were averaged at various phases from the reconstructed image sequence. LVP values were averaged at various phases from the data of 546 cardiac cycles (data, mean ± SEM). The values of SD for peak LVP and HR during measurement were less than ∼5 and ∼1%, respectively. (D) Relationship between SL and LVP during the cardiac cycle. Average values of SL and LVP obtained in B were plotted. Colors indicate the phases in the cardiac cycle (as in right). (E) Histograms showing the SL variances at phases −8 (blue) and 0 (red), i.e., in diastole and systole, respectively. The SL values were 1.74 ± 0.08 µm (643 sarcomeres) and 1.60 ± 0.09 µm (583 sarcomeres) at phases −8 and 0, respectively. A similar magnitude of SD was obtained in both phases. Error bars represent mean ± SEM.
Figure 6.

Cardiac sarcomere imaging with image sequence reconstruction. (A) Data showing the method of image sequence reconstruction for in vivo sarcomere imaging. Shown in the following order from top to bottom (analyzed by PC1; see Fig. 1 A): ECG, LVP, objective lens position (along the Z-axis), and EMCCD camera shutter signal (either “open” or “closed” at 100 Hz). Data obtained 5, 20, and 42 s (from left to right) after the onset of recording (0.5 s at each time point). The objective lens position was moved downward by a piezo actuator at a 1-µm increment along the Z-axis; i.e., 0, 3, and 7 µm at 5-, 20-, and 42-s time points, respectively, after the onset of recording (see B for captured static images). HR, ∼400 bpm in this animal throughout the observation. (B; top) Time course of a change in LVP during the cardiac cycle (divided into 17 phases, from −8 to 8, with a period of one phase at 10.2 ms). (Middle) Images of an AcGFP-expressing myocyte in the LV in vivo captured at phases −8, −2, 0, and 7 at 0, 2, and 6 µm in the Z-direction. (Bottom) Best-focused images shown by asterisks in middle. SL was analyzed by using the multiple Gaussian fitting (see Fig. S4, A–D). SL values were 1.75 ± 0.11 (74), 1.64 ± 0.11 (68), 1.63 ± 0.10 (68), and 1.74 ± 0.10 µm (82) at phases −8, −2, 0, and 7, respectively (number of sarcomeres shown in parentheses). Bar, 20 µm. (C) Time-dependent changes in SL (red) and LVP (black) obtained from the analysis in B using the reconstructed image sequence. SL values were averaged at various phases from the reconstructed image sequence. LVP values were averaged at various phases from the data of 546 cardiac cycles (data, mean ± SEM). The values of SD for peak LVP and HR during measurement were less than ∼5 and ∼1%, respectively. (D) Relationship between SL and LVP during the cardiac cycle. Average values of SL and LVP obtained in B were plotted. Colors indicate the phases in the cardiac cycle (as in right). (E) Histograms showing the SL variances at phases −8 (blue) and 0 (red), i.e., in diastole and systole, respectively. The SL values were 1.74 ± 0.08 µm (643 sarcomeres) and 1.60 ± 0.09 µm (583 sarcomeres) at phases −8 and 0, respectively. A similar magnitude of SD was obtained in both phases. Error bars represent mean ± SEM.

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