Fig. 4.

High-resolution mechanical imaging of zebrafish notochord ECM in-vivo. Brillouin shift maps were acquired in the middle between the left-right animal’s side (posterior, top; anterior, bottom). (A) Confocal image of sheath cells (GFP) as well as corresponding coarse-grained overview Brillouin image in (B) recorded with 1 µm step size at 3 days post fertilization (dpf) centred ~500 µm anterior from the posterior end of the notochord. (C) Brillouin shift map at 3 dpf centred ~250 µm from the posterior end (1 µm step size). (D) High-resolution Brillouin map of boxed area denoted in (C) with a step size of 0.1 µm. (E) Brillouin spectra recorded at discrete positions across the ECM (boxed area in (D)). Double-peak fitting distinguishes two Brillouin shifts of the ECM (orange and blue). Here, the fitting parameters were seeded from the parameters obtained from the fit to the pixel with highest ECM peak contribution and then subsequently constraining the ECM peak around ± 0.7 GHz when analyzing the surrounding pixels in order to prevent artefacts. We note that the observed variability in ECM peak shift is much lower than this fitting constraint. Brillouin shift map for the “high-shift” peak (F) and for the “low-shift” peak (G) – here pixels without a second peak are removed. A spatial map of the ratio of the spectral contributions is plotted in (H). (I) Line plots of ECM contribution to total spectrum across the ECM in (H), centred by their respective maximum. The represented curves are the result of the fitting with the convolution of the measured PSF (Gaussian) and ECM (box function - as shown in the inset). Black line denotes average of individual line plots. Brillouin images were obtained with a 0.85 NA objective and using 8.3 mW of laser power and 0.25 s of exposure time per pixel. w, water; m, muscle; sh, sheath cell; vac, vacuole; ECM, extracellular matrix; d, dorsal; v, ventral. Scale bars, 20 μm in (A,B), 5 μm in (C), 1 μm in (D,F,G,H).