Figure 1.
Figure 1. SL nanometry in α-actinin–AcGFP-expressing rat neonatal cardiomyocytes. (A, top left) Epi-illumination image of a neonatal cardiomyocyte expressed with α-actinin–AcGFP. (left, bottom) Side view of the reconstructed image of the myocyte at the top left. “Top” or “bottom” indicates the direction (i.e., “bottom” attached to the cover glass). See Video 3. (top right) Plot profile of the yellow outlined rectangular region in the image at the top left. (bottom right) Plot profile of the yellow outlined rectangular region in the image at the bottom left. Numbers in plot profiles indicate the values of mean lengths of seven sarcomeres viewed from different angles (NS, P > 0.05 compared with the value at the top right). (B, top left) Epi-illumination image of a neonatal cardiomyocyte expressed with α-actinin–AcGFP in Z discs. Imaging performed at rest; clip from Video 1. The sarcomere in the yellow outlined rectangle was used for the analysis. (bottom left) A graph showing an example of SL nanometry. Fluorescence peak was fitted by a parabolic function of Y = −AX2 + BX − C (A, B, and C are constants) using 3 pixels (i.e., peak and before and after the peak) at each camera frame to obtain the midpoint for the curve. a: A, B, and C, 1,227.7, 3,435.2, and 2,166.0, respectively (X = 3.521 µm). b: A, B, and C, 1,207.1, 8,500.0, and 1,4715.0, respectively (X = 1.399 µm). 1 pixel, 270 nm (for all experiments with neonatal cardiomyocytes). Because of the mathematical nature, the parabolic function passes all 3 pixels. The midpoint of the parabolic function was defined as the Z-disc position, and SL was derived by the difference between the adjacent two midpoint values. (top right) A three-dimensional graph showing changes in the positions of a and b from frame 1 (same as in bottom left) to 6. Yellow lines, time-dependent changes in the Z disc. (middle right) Kymographs showing changes in the longitudinal positions of the AcGFP fluorescence signals. a and b indicate the AcGFP fluorescence signals in the yellow outlined rectangle in the top left image. Time course, 2 s (as shown by dashed arrows). The time period shown by a red rectangle was used for the analysis in the top right. (bottom right) Fluctuation of the length of a single sarcomere (distance between a and b). SD (i.e., SL displacement precision) was 3.37 nm. a.u., arbitrary units.

SL nanometry in α-actinin–AcGFP-expressing rat neonatal cardiomyocytes. (A, top left) Epi-illumination image of a neonatal cardiomyocyte expressed with α-actinin–AcGFP. (left, bottom) Side view of the reconstructed image of the myocyte at the top left. “Top” or “bottom” indicates the direction (i.e., “bottom” attached to the cover glass). See Video 3. (top right) Plot profile of the yellow outlined rectangular region in the image at the top left. (bottom right) Plot profile of the yellow outlined rectangular region in the image at the bottom left. Numbers in plot profiles indicate the values of mean lengths of seven sarcomeres viewed from different angles (NS, P > 0.05 compared with the value at the top right). (B, top left) Epi-illumination image of a neonatal cardiomyocyte expressed with α-actinin–AcGFP in Z discs. Imaging performed at rest; clip from Video 1. The sarcomere in the yellow outlined rectangle was used for the analysis. (bottom left) A graph showing an example of SL nanometry. Fluorescence peak was fitted by a parabolic function of Y = −AX2 + BX − C (A, B, and C are constants) using 3 pixels (i.e., peak and before and after the peak) at each camera frame to obtain the midpoint for the curve. a: A, B, and C, 1,227.7, 3,435.2, and 2,166.0, respectively (X = 3.521 µm). b: A, B, and C, 1,207.1, 8,500.0, and 1,4715.0, respectively (X = 1.399 µm). 1 pixel, 270 nm (for all experiments with neonatal cardiomyocytes). Because of the mathematical nature, the parabolic function passes all 3 pixels. The midpoint of the parabolic function was defined as the Z-disc position, and SL was derived by the difference between the adjacent two midpoint values. (top right) A three-dimensional graph showing changes in the positions of a and b from frame 1 (same as in bottom left) to 6. Yellow lines, time-dependent changes in the Z disc. (middle right) Kymographs showing changes in the longitudinal positions of the AcGFP fluorescence signals. a and b indicate the AcGFP fluorescence signals in the yellow outlined rectangle in the top left image. Time course, 2 s (as shown by dashed arrows). The time period shown by a red rectangle was used for the analysis in the top right. (bottom right) Fluctuation of the length of a single sarcomere (distance between a and b). SD (i.e., SL displacement precision) was 3.37 nm. a.u., arbitrary units.

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