Fig. 3: SeeThrough enables minimally invasive brain imaging.

a Schematic of the experimental design. b–f Calibration curves (black lines) for each chemical to determine its concentration in the brain parenchyma (b EDTA; c SDBA; d EtOH; e urea; f ANP). Black squares indicate signals measured by standard concentrations for each chemical (standard concentrations: b 3.125, 6.25, 12.5 µM; c 0.001, 0.002, 0.005, 0.008%; d 0.005, 0.010, 0.020, 0.025%; e 0, 1.25, 5, 10, 12.5 mg/dl; f 1, 2, 5, 10 µM). Blue triangles and red circles indicate the determined concentrations in control (PBS-treated) and SeeThrough-treated samples (n = 3), respectively (PBS: d 0.015%; e 7.54 mg/dl; SeeThrough: d 0.007%; e 6.63 mg/dl; f <1 µM). Detection kits (b–e) and LC-MS (f) were used for the quantification assay. Data were presented as mean ± SEM. g Monitoring tdTomato-expressing microglia in the visual cortex in Iba1-tdTomato transgenic mice 0 h (left) and 24 h (right) after the SeeThrough treatment (top), and the open-skull surgery with (w/, middle) or without (w/o, bottom) durotomy. h Comparison of the tdTomato fluorescence between 0 and 24 h after the SeeThrough treatment (left, n = 9 areas from three mice, P = 0.5703, two-tailed Wilcoxon test) and open-skull surgery with (middle, n = 9, **P = 0.0039, two-tailed Wilcoxon test) or without (left, n = 7 from three mice, P = 0.2968, two-tailed Wilcoxon test) durotomy. ns not significant. Data were presented as mean ± SEM. i Immunofluorescent images of the visual cortex in the coronal brain sections using anti-GFAP antibody. Brains were fixed without any treatment (left), 24 h after the SeeThrough treatment (second left), and 24 h after the open-skull surgery with (middle) or without durotomy (second right). To assess the long-term effect, SeeThrough was performed three times over seven days (on the first, fourth and seventh days) and then the brains were fixed (right). Cortical regions just below the treated areas were examined. j Comparison of the GFAP immunoreactivity among no treatment (n = 4 areas from two mice), 24 h after the SeeThrough treatment (n = 6 from three mice), 24 h after the open-skull surgery without or with durotomy (n = 6 from three mice for each), and 7 d after the initial SeeThrough treatment (n = 12 from three mice). *P = 0.0275 (no treatment vs open-skull with durotomy), 0.0305 (open-skull with durotomy vs SeeThrough, 7 d), and 0.0493 (open-skull with durotomy vs open-skull without durotomy), **P = 0.0029, two-tailed Kruskal–Wallis test. Data were presented as mean ± SEM. k Schematic of the experimental design. l Measurement of the bone mechanical strength. m Mechanical strength of parietal bone samples surface-treated for ten sessions with PBS or SeeThrough solutions, and whole-immersed for ten sessions with SeeThrough solutions (n = 7 for surface immersion with PBS and n = 9 for surface and whole immersion with SeeThrough solutions, **P = 0.0038, ***P = 0.0008, one-way ANOVA with Tukey’s multiple comparison test). Data were presented as mean ± SEM. Scale bars, 100 μm. Credits. Panel a include illustration elements © Shutterstock.com (licensed) and panel k include illustration elements © ACworks/illustAC, not covered by the article’s CC‑BY 4.0 licence.