Fig. 2

Effects of focal depth on the fluorescent image. A, B Same fluorescence image was obtained when the focal plane was set near the surface. This focal depth was set at 0 μm. Regions with higher fluorescence intensity are shown in darker colors in these fluorescence images. At this focal plane, many elliptical regions darker than the peripheral regions were visible. Each dark elliptical region was regarded as a single cell, and the outline of each cell was selected using the ellipse tool (A). A total of 38 regions were selected and classified into three groups based on their amplitudes of inspiratory-related fluorescence changes: strong (n = 15), thick black line; weak (n = 10), thin black line; and no (n = 13), thick white line inspiratory-modulated groups (see the Results for the group definitions). Raw and averaged fluorescence signals in regions 1 to 6 (labeled R1 to R6 in panel A) are shown in Figs. 4 and 5. C–G Fluorescence images obtained from the same area in panel B, when the focal depth was 10, 20, 30, 40, and 50 μm deeper than the focal plane in panel B, respectively