Simultaneous measurement of single SL and [Ca²⁺]_i in neonatal myocytes during spontaneous beating. (A, top) Epi-illumination image of an α-actinin–AcGFP-expressing myocyte loaded with Fluo-4. Numbers shown by arrows indicate sarcomeres used for the analyses. (middle) Time course of changes in the lengths of seven sarcomeres and the FI of Fluo-4. (bottom) Superimposed data showing SL changes. Six consecutive beats are shown. See Video 6. (B, top) Kymograph showing changes in the lengths of the seven sarcomeres (second and third beats; compare with A). Color bar at right indicates the pseudo-color representation of FI (arbitrary). (bottom) Graph showing changes in the lengths of the seven sarcomeres and the FI of Fluo-4 (second and third beats). Individual SL data (corresponding to the sarcomeres numbered with different colors in A), averaged SL data (black line), and [Ca²⁺]_i changes (red line) are shown on the same time course. (C, top) Bar graphs comparing the values of shortening time (left) and shortening velocity (right) obtained by using different types of analysis. (bottom) Bar graphs comparing the values of lengthening time (left) and lengthening velocity (right) by using different types of analysis. Single SL (averaged individual SL, from 1 to 7, during six consecutive beatings) versus averaged SL (from 1 to 7 during six consecutive beatings). *, P < 0.05. Error bars indicate means ± SEM. a.u., arbitrary units.