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

Figure 3

Visualization of cardiomyocytes by CellMask in the LV in vivo. (a) Confocal images showing myocytes as well as capillaries in two different regions (i.e., Regions #1 and #2) in the LV of a mouse. CellMask was injected into the LV chamber via the apex of the heart. Region #1 is at the center of the LV, and Region #2 is ~3 mm closer to the apex of the heart. Note marked FI in capillaries in both regions. Arrowheads indicate gap junctions. Bar = 20 μm. In both images: observed with a 40× lens (exposure time, 28 ms). (b) Same as in (a) in the LV of a mouse, but CellMask (0.5 μg/mL, 10 μL) was directly injected into the epicardial surface. Arrow indicates the injected region showing fragmented cells. Gap junctions are not clearly observed by this method. Bar = 40 μm. Observed with a 20× lens (exposure time, 28 ms). (c) Illustration showing the CellMask treatment on the LV epicardial surface. A small piece of gauze (~5 × 5 mm²) was gently placed on the LV surface and the CellMask solution (0.1 μg/mL, ~1 mL) was dropped onto it for ~5 min using a pipette. The animal, anesthetized with 2% isoflurane, was ventilated during the CellMask treatment. (d) Top: confocal image showing myocytes in the instillation region in the LV of a mouse. Arrowhead indicates a gap junction. Bar = 20 μm. Observed with a 60× lens (exposure time, 9.8 ms). Bottom: FI profile in the yellow-rectangular regions in Cells #1 and #2 in Top. T-tubular distance, 2.42±1.17 and 2.26±1.00 μm in Cells #1 and #2, respectively. (e) Top: same as in (d) Top via superposition of 25 images [the same myocytes as in (d) Top are shown]. Arrowhead indicates a gap junction. Bar = 20 μm. Bottom: FI profile in the yellow-rectangular regions in Cells #1 and #2 in Top. T-tubular distance, 2.24±0.05 and 2.10±0.23 μm (P=0.15) in Cells #1 and #2, respectively. The T-tubules are numbered in the image, and “1” and “10” in FI profiles indicate fitted fluorescence signals for the corresponding T-tubules.

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