2.1.

Preparation of Brain Sections and Clearing Agents

Adult wild-type mice (C57BL/6J) and Thy1-green fluorescent protein (GFP) mice (line M, Jackson Laboratory) were used in this study. Mice were anesthetized with a mixture of 2% $\alpha$-chloralose and 10% urethane ($8\mathrm{mL}/\mathrm{kg}$) through intraperitoneal injection, and perfused intracardially with 0.01 M PBS (Sigma) followed by 4% PFA (Sigma). The brains were postfixed overnight at 4°C in 4% PFA. The mouse brains were rinsed with PBS several times and were sliced into $100\text{-}\mu \mathrm{m}$ coronal sections with a vibratome (Leica VT 1000s). For prodium iodide (PI) staining, which is widely used in fluorescence microscopy as a popular red-fluorescent nuclear labeling dye, the sections were incubated in PI dilution ($2\mu \mathrm{g}/\mathrm{ml}$) for 1 h before clearing. Animal care and experimental protocols were approved by the Experimental Animal Management Ordinance of Hubei Province, P. R. China.

MD simulation was used to predict the optical clearing potential of the agents. The detailed protocols were described in our previous work.³⁶ After the simulation, the number of hydrogen bonds between each agent and the peptide was counted every 1 ps during the entire simulation, and their sum was calculated as the total number. The average number of hydrogen bonds was obtained by dividing the total number by the production run time as well as by the number of molecules.³⁶

The three saturated solutions at 20°C, sorbitol, sucrose, and fructose, were used to clear brain sections with simple immersion or topical treatment. Eighty percent glycerol, which is a common mounting medium, was also used here. ${\text{Clear}}^{\mathrm{T}2}$ was a relatively rapid method for neuronal and non-neuronal tissue, which needs three steps to make the tissue transparent.¹⁴ Here, we used the higher concentration mixture (50% formamide and 20% PEG8000) in ${\text{Clear}}^{\mathrm{T}2}$ to compare the fluorescence preservation and imaging depth with the above agents. The viscosities of agents were measured respectively with the viscometer (NDJ-5S, JNT, Shanghai, China) at room temperature. The concentration and viscosity of all the agents are listed in Table 1.

Table 1

The concentration and viscosity of clearing agents used in this study.

2.2.

Microscopy and Imaging

Before and after treatment with different agents, the samples were put on a 1951 United States Air Force (USAF) resolution test target. Due to the inhomogeneity of brain tissue, the middle region (striatum) of the brain section was put on the central area of 1951 USAF target. White-light images were taken by a CCD camera (PixelFly USB, PCO computer, Germany) attached to a stereo microscope (SZ61TR, Olympus, Japan). The brain sections were mounted with two cover glasses for fluorescence imaging. The fluorescence images were acquired with confocal fluorescence microscopy (LSM710, Zeiss, Germany) equipped with the Plan-Apochromat $20\times /0.8$ dry objective (W.D. 0.55 mm).

2.3.

Quantification Analysis

The obtained images were analyzed using ImageJ software and calculated with MATLAB.

For fluorescence quantification, the elliptical-selection tool was used to select an elliptical area in the soma of a neuron, and the histogram tool was used to measure the mean fluorescence intensity of the ellipse area, which served as the fluorescence intensity of the neuron. The fluorescence intensity of a neuron is supposed to be “A” before clearing and “B” after clearing. We calculated the fluorescence change of a neuron during clearing as “B/A.” For each group, a mean value of fluorescence change of 10 neurons was calculated.

For imaging depth, the decay of image contrast value with depth in brain slice before and after clearing was used for quantification. The imaging contrast was calculated with Eq. (1), where $I$ is the grayscale value for each pixel, $I_{\text{mean}}$ is the average intensity of the image, and $n$ is the number of total pixels. The imaging depth was determined at the point where the contrast value had fallen to $1/e$ of the maximum contrast at the tissue surface. The imaging depth after clearing was divided by the depth before clearing, which served as the increase of imaging depth for different agents.

For size change measurement, the length of straight lines connecting the feature points of the images from dorsal to ventral and from lateral to middle were measured, and served as deformation in the longitudinal and horizontal directions, respectively. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test.

3.1.

Transparency of Samples with Different Agents

In this study, the optical clearing potential of some commonly used chemical agents with rich hydroxyl groups was investigated with MD simulation, including sorbitol, sucrose, fructose, and glycerol. The propensity to form hydrogen bonds was employed to predict the clearing potential,^36,39–40 as shown in Table 2.

Table 2

Numbers of hydrogen bonds of several agents with MD simulation.

The simulation results show that sorbitol, sucrose, and fructose form more bonds than glycerol. Therefore, it can be deduced that the former three agents have stronger ability to disrupt the hydration shell around the collagen and better optical clearing potentials.

Figure 1 shows the white-light images of $100\text{-}\mu \mathrm{m}$-thickness brain sections to demonstrate the rapid clearing process within 2 min. The results show that the striatum regions of the brain, which have abundant nerve fiber, become transparent rapidly by simple immersion or topical treatment within 2 min with sorbitol, sucrose, fructose, and glycerol. After the 2-min treatment, the resolution of the 1951 USAF target measured through brain slice is $16.31\pm 1.12\mu \mathrm{m}$, $16.95\pm 1.12\mu \mathrm{m}$, $14.81\pm 1.21\mu \mathrm{m}$, and $30.18\pm 1.97\mu \mathrm{m}$ for sorbitol, sucrose, fructose, and glycerol, respectively. This is to say that the former three agents can achieve better transparency than the latter one, and fructose presents the best clearing effect.

Fig. 1

White-light images of brain sections before and after clearing with different agents. The background is the central area of 1951 USAF.

3.2.

Increase of Imaging Depth with Different Agents

To investigate the improvement of imaging depth for different agents, the GFP fluorescence images of brain samples before and after clearing were acquired with same parameter settings with confocal microscopy.

Figure 2(a) shows the maximum projections of the transverse ($x-y$) and orthogonal ($x-z$) image stacks by taking sorbitol as an example. The $x-z$ projection images indicate the increased imaging depth after clearing.

Fig. 2

(a) Maximum projections of transverse ($x-y$) and orthogonal ($x-z$) image stacks. (b) Increase of imaging depth with different agents. Scale bar, $50\mu \mathrm{m}$. *, significant ($P<0.05$).

To quantify the improvement of imaging depth induced by the agents, the contrast of the images was calculated according to Eq. (1), and the imaging depth was obtained based on the contrast decay, as shown in Fig. 2(b). The results show that the imaging depth has been increased almost threefold for sorbitol, sucrose, and fructose, which is higher than ${\text{Clear}}^{\mathrm{T}2}$ with significant differences. In other words, treatment with the three clearing agents can significantly improve the optical imaging depth.

3.3.

Fluorescence Preservation of GFP and Prodium Iodide with Different Agents

The genetically encoded fluorescent proteins, such as GFP, are important biomarkers for visualizing both structural and functional details in tissue. Thus, it is particularly important to keep an eye on the fluorescence preservation of the endogenous proteins.

To study the influence of the three agents on GFP fluorescence, the images of brain sections were acquired with same parameter settings with confocal microscopy before and after clearing. As shown in Fig. 3(a), the GFP fluorescence is preserved well for all the agents, including sorbitol, sucrose, fructose, and ${\text{Clear}}^{\mathrm{T}2}$. From the images of the maximum projection of the same thickness, it can be seen that the fluorescent signals in deep layers of the tissue became stronger, and some new signals in deeper tissues could be obtained after clearing. The quantitative results shown in Fig. 3(b) shows that sorbitol and sucrose evidently enhance the mean fluorescence of GFP as well as ${\text{Clear}}^{\mathrm{T}2}$, while fructose keeps the mean value almost as before clearing. Hence, sorbitol and sucrose could preserve GFP fluorescence better than fructose.

Fig. 3

Fluorescence preservation of GFP and PI. (a) Fluorescence images of GFP labeled neurons. (b) Mean fluorescence change of GFP treated with different agents. (c) Fluorescence images of PI stained cells. (d) Mean fluorescence change of PI treated with different agents. Scale bar, $50\mu \mathrm{m}$. Each image is a maximum projection of the $z$-stacks (thickness: $30\mu \mathrm{m}$).

It is also examined whether the clearing agents are compatible with PI, which is a common nuclear staining dye in biological research.

Figure 3(c) shows the fluorescence images of PI stained brain slices cleared with different agents. It can be seen that the fluorescence intensity for sorbitol, sucrose, and fructose preserve well, while for ${\text{Clear}}^{\mathrm{T}2}$, fluorescence quenching is obvious. For further analysis, we quantified the fluorescence preservation of the PI signal with ImageJ software as described earlier. As shown in Fig. 3(d), the mean fluorescence intensity of PI stained cells for sorbitol, sucrose, and fructose increase by 1.5, 2.3, and 3 times, respectively, while ${\text{Clear}}^{\mathrm{T}2}$ results in a 58% loss of PI fluorescence intensity.

In general, sorbitol and sucrose evidently increase both GFP and PI fluorescence intensity. Fructose can greatly increase the fluorescence intensity of PI, while slightly decreasing the fluorescence intensity of GFP.

In addition, it can be seen from the images in Figs. 3(a) and 3(c) that the visual fields after clearing for sorbitol, sucrose, and fructose are larger than before, which shows sample shrinkage. This might be induced by dehydration due to the hyperosmosis and high concentration of these agents.

3.4.

Tissue Size and Cell Morphology with Different Agents

During the clearing process, the changes of sample size are inevitable due to the dehydration or hydration of different agents. Cell morphology preservation is of particular importance to analyze fine structures of biological tissues. The consistency of the contraction or expansion rate in different directions of the sample determines the basic structure preservation.

The contraction rates after clearing in the horizontal and longitudinal directions were calculated, respectively. As shown in Fig. 4(a), for the three clearing agents, the contraction rates in the horizontal and longitudinal directions are very close, and there is no significant difference between the two directions. In the case of ${\text{Clear}}^{\mathrm{T}2}$, the horizontal contraction is significantly smaller than longitudinal, and this inconsistent contraction in the two directions will lead to morphologic deformation. Tissue contraction is more beneficial for morphology preservation since swelling might induce structure rupture.

Fig. 4

(a) Contraction rates of samples in two directions of different agents. Horizontal indicates the direction from left to right, and longitudinal indicates the direction from dorsal to ventral. N.S., non-significant ($P>0.05$). *, significant ($P<0.05$). (b) Morphology preservation of pyramidal neurons (cortex) before and after optical clearing with sorbitol solution. Scale bar, $20\mu \mathrm{m}$. Each image is a maximum projection of $z$ stacks (thickness: $40\mu \mathrm{m}$).

To evaluate the cell morphology preservation, we imaged the pyramidal neurons in the cortex in $100\text{-}\mu \mathrm{m}$ tissue sections before and after clearing with the agents. After clearing with sorbitol, the neuronal morphology remained intact, as shown in Fig. 4(b). Sucrose and fructose show fine structure preservation as well as sorbitol (data not shown).