The applicability of SHG measurements for evaluation of sarcomeric A-band 3-D structure. (A) A series of five SHG images of A-bands in an isolated cardiomyocyte obtained at different positions along the Z-axis, i.e., from the top (slice #1) to the bottom of the cell (slice #5). The yellow-colored ROI on each frame is kept fixed in size and X/Y positions and used to retrieve A-band axial regularity. Scale bar on the top image is common for all images. (B) Curves of A-band regularity patterns obtained from the corresponding ROIs shown in the images in A. (C) The shifts of individual A-band X position across the slices as calculated relative to the X-position in the center of the cell (i.e., slice #3). The plots are obtained for four frames (two below and two above the middle frame). The magnitude of the longitudinal shifts, their variation along the x axis, and the actual direction of the change during the axial slicing across the cell depth may be used to estimate z-axis A-band (mis)alignment.