Fig. 1: Intervertebral window of retaining ligamentum flavum.
From: Long-term in vivo imaging of mouse spinal cord through an optically cleared intervertebral window
a Schematic diagram of the intervertebral window with ligamentum flavum (LF). b Cross-sectional schematics of the LF window. c, d Projection and 3D reconstruction of an in vivo multimodal image stack of the LF window. Green: GFP labeled microglia; Red: blood vessels labeled with Texas Red dextran; Gray: second harmonic generation (SHG) signals of collagen and other connective tissues; Magenta: stimulated Raman scattering (SRS) signals of adipose tissue and myelin with the Raman shift at 2863.5 cm−1, attributed to the vibration of the methylene group enriched in lipids. Scale bar, 500 \(\mu\)m. e Maximal projection of the image volume between the two planes of depth from 300–500 \(\mu\)m indicated in (d) showing the distribution of blood vessels and adipocytes in the epidural space. Cells labeled by Texas Red in the epidural space are probably invading immune cells. Cells shown in the inset with strong pump-probe absorption at 2863.5 cm−1 are red blood cells indicated by their specific dumbbell shape. f Two-photon fluorescence image of a 50-\(\mu\)m-thick longitudinal spinal cord slice under the LF window. Scale bar, 50 \(\mu\)m. g, h Evaluation of the microglial ramification index (g) and number of process endpoints (h) of spinal cord fixed slices from the LF window group, the dorsal column crush (DCC) group and the negative control (N.C.) group. n \(\ge\) 20 microglial cells were analyzed from 6–8 slices per mouse, three mice per group. i The in vivo superimposed images of microglia at 1 hour and 3 hours post-surgery, showing ramified microglia with highly motile processes under the LF window. Scale bar: 50 \(\mu\)m. j, k Changes of the microglia ramification index (j) and number of process endpoints (k) during two-hour in vivo imaging in the LF window group and the DCC group. Images of the same region were taken at an interval of 30 minutes. 6–8 microglial cells in the same imaged region were analyzed per time point per mouse. Error bars, s.e.m. l, m In vivo evaluation of the microglial ramification index (l) and process endpoints (m) of ten mice with LF window at the first live imaging session. The in vivo morphological indices from the three non-activated mice with LF (LF#1–3 in (j–k)) were used as the gold standard (G.S.) for in vivo microglia activation evaluation. The in vivo results from the DCC group were used as the positive control. Microglia activation in each mice was determined by comparing the calculated ramification index and number of process endpoints with the G.S. 7–12 microglial cells were analyzed for morphological quantification for each mouse; the box plots are shown with median, upper and lower quartiles and max and min values. Kruskal-Wallis test: ****P \( < \) 0.0001. Data of the DCC and N.C. groups are shared with Supplementary Fig. 3. Images shown in (f) and (i) are representative results of three experiments in three mice. Figure (a) created using BioRender (https://biorender.com/). Source data are provided as a Source Data file.
