MIMS analysis of stereocilia from mice fed 15N-L-leucine. (a-f) Quantitative MIMS images of cochlear hair cells from mice after 9 days on the 15N-L-leucine diet. DC, Deiter cells; OP, outer pillar cells; RL, reticular lamina; TBC, tympanic border cells (below the basilar membrane); Sb1 and Sb2, stereocilia bundles; other abbreviations are as in Figure 2. All images are 60 × 60 μm (256 × 256 pixels) and have an acquisition time of 10 msec/pixel. (a) 12C14N-, (b) 12C15N-, (c) 12C15N-/12C14N- ratio image, (d) 12C-, (e) 13C-, (f) 13C-/12C- ratio image. The images in (c,f) result from the pixel-by-pixel division of the 12C15N- image by the 12C14N- image and of the 13C- image by the 12C- image, respectively. Scale bar = 10 μm. (g-l) High-resolution quantitative MIMS images of the stereocilia labeled Sb1 in (a). The isotopes and ratios shown in each image are indicated and are the same as the equivalent images in (a-f). All images are 3 × 3 μm (256 × 256 pixels) and an acquisition time of 40 msec/pixel. Scale bar = 0.5 μm. (m) HSI image of the 12C15N/12C14N ratio derived from (h) and (g). The colors correspond to the excess 15N derived from the measured 12C15N-/12C14N- isotope ratios, expressed as a percentage of the 15N excess in the feed, which is a measure of protein renewal; values range from 0% (blue) to 60% and higher (magenta). Small magenta areas (α, β, γ, δ, ε, and ζ) indicate excess 15N. The image is 3 × 3 μm (256 × 256 pixels) and dwell time was 40 msec/pixel. (n) Bar graph of the mean percentage at the stereocilia level of the 15N excess in the feed, which is a measure of protein renewal, after 9 days or 22 days of 15N-L-leucine diet. L, inter-stereocilia structures; S, core stereocilia at 100–200 nm from L. (o) Bar graph of the mean value of the 13C/12C ratio measured after 9 days at the same locations as in (n). t, value of the natural terrestrial 13C/12C ratio.