Measurement of tissue size change. The measurements of sample linear expansion or shrinkage were based on the bright-field images. The brain was outlined to measure the area using the “polygon-selections” function of ImageJ software. The linear size change value was quantified by the square root of area size change (Fig. 3D).

Imaging depth quantification. The z depth of LSFM images supposed to be “d” was measured from the surface to the inner part of the brain until the last identifiable cell over the background. The length of the cleared brain (dorsal to ventral) was supposed to be “D”. The imaging depth was quantified as “d/D” (Fig. 3F).

Normalized mean fluorescence quantification. To assess the fluorescence preservation of the clearing methods, we quantitatively calculated the normalized mean fluorescence intensity of fluorescence images at the indicated time points during clearing. For each brain sample at each time point, the MIP of the image stack (40 to 60 μm thick) was acquired from the surface of the brain slice in the cortex. Next, the signal and background area were separated from each z projection image by a threshold function. As the intensity of the signal area includes the signal intensity and the background intensity (26), the mean fluorescence intensity M of an image was calculated as followsEmbedded Image(1)I and N are the sum of the pixel intensity and pixel number, respectively, of the signal or background. The mean fluorescence intensity of an image is supposed to be “Mb” before clearing, “M0” at 0 day after clearing, and “Mi” at i day during long-term storage. The fluorescence intensity preservation during clearing (0 day) was normalized to the intensity before clearing as “M0/Mb” [Figs. 1G and 2 (C and I) and figs. S1 (B and D), S2B, and S7B], and the fluorescence preservation at each time point (i day) during long-term storage was normalized to the intensity at 0 day after clearing as “Mi/M0” [Figs. 1I and 2 (E and J) and figs. S1D and S2D]. The fluorescence preservation of recombinant EGFP in the brain samples was quantitatively calculated during long-term storage (Fig. 1B).

Fluorescence level quantification. The fluorescence level (Figs. 3E and 4D) was expressed as the signal-to-background ratio and quantified as described in the uDISCO paper (26). From each brain scan, a 100-μm MIP of image stacks was acquired at the same cortex region. To calculate the mean intensity of the signal for each z projection, we used the threshold function of ImageJ software to separate the signal from the background and then measured the mean intensity of the signal by the “analyze particles” function. The mean intensity of the background was obtained by averaging the values of five regions without a signal in the z projection. The fluorescence level value was calculated by dividing the mean signal intensity above the background.

Normalized signal profile. In the ImageJ software, a straight line (length, 200 μm) was drawn across the specific signal, and the fluorescence signal was measured by the “plot profile” function. The intensity of the background was measured as the value of the baseline. Next, the fluorescence signal profile was normalized by the background intensity (fig. S5C).

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