Fig. 1: Development of the RI matching solution suitable for in vivo skull clearing.
From: SeeThrough: a rationally designed skull clearing technique for in vivo brain imaging
a Schematic workflow for the development of woRIMS. To develop a skull clearing protocol that achieves both high RI (RI = 1.56) and biocompatibility, an aqueous intermediate solvent (AqIS) was employed, along with a mixture of a wRIMS and an oRIMS that is miscible with the AqIS. dD dispersion parameter, dP polar parameter, dH hydrogen-bonding parameter, eRI estimated RI. b A plot of dP and dH distances between a selected set of 20 organic solvents and a 50–75% ethanol aqueous solution. Filled and open circles indicate “miscible” and “immiscible,” respectively. The RIs of each organic solvent were displayed using the indicated color code. In a 50–75% ethanol aqueous solution, the miscible radius of the HSP sphere is ~20, indicated by the red curve. c Hydration scores for 640 independent, non-detergent, and salt-free chemicals re-analyzed from ref. 11 (Supplementary Fig. 2 and Supplementary Data 2) are displayed as box-and-whisker plots (boxes, 25–75 %; whiskers, 10–90 %; vertical line, median; cross, mean); for each chemical the value plotted is the average of two technical replicates, whereas reference compounds (C#0001–0012) used for normalization are excluded from n. Normality (Kolmogorov–Smirnov, α = 0.05) failed only for the alcohol group; therefore, functional groups were compared with the alcohol group by a two-sided Steel test (family-wise α = 0.05). Exact results for significant increases: 1° amine (T = 6.76, ρ = 0.194, P = 1.88 × 10−10), 3° amine (T = 4.13, P = 5.05 × 10−4), urea (T = 4.41, P = 1.47 × 10−4), nitrile (T = 4.74, P = 3.00 × 10−5), aromatic (T = 5.79, P = 9.61 × 10−8), phenyl (T = 3.99, P = 9.02 × 10−4), and pyridine (T = 5.08, P = 5.41 × 10−6); all other groups showed P > 0.05. The median estimated refractive index (eRI) for each chemical group is indicated by color coding. d A plot of RI values for saturated mixed solutions of the selected wRIMS candidates with BA (black square) or VA (red circle) against the HSP distance between those wRIMS and BA or VA (n = 3). The RIs of BA and VA are indicated by the black and red lines, respectively. Data were presented as mean ± SD. e The visual appearance of the BA/#0640 and BA/#1050 mixture. f Bright-field images of the isolated skull before and after clearing, and a plot of quantified transmittance after clearing. Data were shown as mean ± SD (n = 3 independent mice per treatment, one skull per mouse, ***P < 0.001, ns: P > 0.9999, one-way ANOVA with Tukey’s multiple comparison test. P < 0.0001 for PBS + PBS, AqIS + SDBA, AqIS + glycerol, EDTA + glycerol, and AqIS + RIMS-1.51, and P = 0.0008 for AqIS + VA/ANP, when compared with AqIS + BA/ANP). Each solution was referenced from past literature; *1: ref. 7, *2: ref. 6, *3: ref. 8, *4: ref. 10. g The chemical structures of benzyl alcohol and antipyrine, and the solution composition of woRIMS. Scale bar, 2 mm (f). Credits. Panel a includes illustration elements © Shutterstock.com (licensed), not covered by the article’s CC‑BY 4.0 licence.
