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. 2016 Sep 20;111(6):1295–1303. doi: 10.1016/j.bpj.2016.08.013

Figure 2.

Figure 2

Physical dimensions of sarcomeres evaluated by optical microscopy. (A) Phase-contrast (top) and confocal fluorescence (middle and bottom) images of myofibrils prepared from glycerinated flight and leg muscles in a relaxing condition. Actin filaments were stained with Alexa Fluor 488 phalloidin after formaldehyde fixation. Myosin filaments were stained with TMR-maleimide. Yellow arrows indicate the Z-lines. (B) Sarcomere length and A-band width (myosin filament length) measured in phase-contrast images. Left: a representative image and corresponding intensity profile are shown. The sarcomere length was defined as the distance between the Z-lines indicated by black arrows (marked as S.L. and vertical lines). Another pair of vertical lines (marked as A-band) indicate the midpoints of fluorescence intensity between the highest and lowest intensity values next to the Z-lines. Right: summary of sarcomere lengths and A-bands determined by phase-contrast images. (C) Left, top: sarcomere length and I-Z-I brush width (twice that of the thin-filament length) measured from Alexa 488-phalloidin images. The sarcomere length was defined as the distance between the Z-lines indicated by black arrows (marked as S.L. and vertical lines). Another pair of vertical lines (marked as I-Z-I) indicate the midpoints of fluorescence intensity between the points where the intensity begins to decline and the lowest-intensity values. Left, bottom: A-band width measurement in TMR-maleimide images. A pair of vertical lines (marked as A-band) indicate the midpoints of fluorescence intensity between the lowest- and highest-intensity values next to the Z-lines (black arrows). Right: summary of sarcomere lengths and lengths of the thin filament (half of the I-Z-I brush width) and thick filament (A-band) obtained from confocal fluorescence images. Means and standard deviations are summarized in Table S1. Scale bars, 5 μm. NS, not significant. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Values of the significance tests are summarized in Table S2. To see this figure in color, go online.