Optimization of Fluorescent Labeling for In Vivo Nanoimaging of Sarcomeres in the Mouse Heart

Figure 1

Expression of -actinin-AcGFP in cardiomyocytes in mice. (a) Top, confocal image showing ventricular myocytes expressed with α-actinin-AcGFP by an ADV injection of 10 μL at 2 × 10¹¹ VP/mL. Values on ADV shown in the upper right corner. Middle: enlarged view of the region of interest (i.e., yellow outlined rectangle) in the confocal image in Top. Bottom: FI profile of the confocal image. Closed circles, plot profile of the image (raw data). Solid line, data analyzed by the multipeak Gaussian fitting. Numbers in the graph indicate the Z-disks in the confocal image. Average distances between FI peaks (i.e., SL), 1.92±0.08 μm. Note that this method collectively derives all of the SL (or T-tubular distance; see Figure 3(e)) values along the region of interest. (b) Same as in (a) by an ADV injection of 10 μL at 4 × 10¹¹ VP/mL. Average length of 9 sarcomeres, 2.10±0.09 μm. (c) Same as in (a) by an ADV injection of 10 μL at 2 × 10¹² VP/mL. Average length of 9 sarcomeres, 1.34±0.12 μm. In (a)–(c): Top, Bar = 20 μm. Middle, Bar = 5 μm. The Z-disks are numbered in confocal images, and “1” and “10” in FI profiles indicate fitted fluorescence signals for the corresponding Z-disks. Observed with a 60× lens (exposure time, 28 ms). See Table 1 for summarized SL data. (d) Top: same as in (a) by an ADV injection of 30 μL at 4 × 10¹¹ VP/mL. Bar = 20 μm. Middle: enlarged view of the region of interest (i.e., yellow outlined rectangle) in the confocal image in Top. Bar = 5 μm. Length of 8 sarcomeres, 1.52±0.44 μm. P<0.05 compared with the value in (a) or (b). Observed with a 60× lens (exposure time, 28 ms). Note disordered striation patterns in myocytes. (e) Confocal image of the ADV-injected area in the LV surface in (d). Bar = 500 μm. Observed with a 2× lens (exposure time, 28 ms). (f) Confocal image of mesothelial cells [just above the first layer of cardiomyocytes, i.e., shown in (d)]. Note the clear fluorescence signal in some mesothelial cells (shown by arrows). Bar = 20 μm. Observed with a 60× lens (exposure time, 28 ms).

(a)

(b)

(c)

(d)

(e)

(f)