Figure 8.

Spatial resolution of the imaging system in living heart tissue. (A) Series of X, Y scans obtained from an isolated perfused mouse heart loaded with rhod-2 using TPLSM in non-descanned mode. Images were collected across a bifurcating capillary at 2-µm z-steps intervals during continuous electrical point stimulation at 4 Hz. Numbers indicate imaging depth (in µm) in reference to the image in panel a. The wave length of excitation light was 810 nm and rhod-2 fluorescence was collected between 560 nm and 650 nm. Green lines in panel c mark the capillary endothelium. Asterisks denote endothelial cell nuclei. Periodic increases in rhod-2 fluorescence resulting from action potential–evoked increases in cytosolic calcium concentration are visible as ripple-like wave fronts. Some X, Y scans reveal the appearance of cardiomyocyte-typical rhod-2 transients within the perfusate-filled capillary lumen (arrows). Scale bar, 10 µm. (B) Data sets shown in A were converted to a stack, and the stack was then resliced to obtain X, Z projections of the capillary. Red lines in the X, Y scan on the left indicate the X positions of the X, Z profiles shown on the right. Green arrows denote action potential-induced rhod-2 transients. In all three projections, rhod-2 transients are clearly detectable within the capillary lumen. Scale bars: 10 µm laterally, 5 µm axially.