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. 2022 Jun 19;23(12):6826.
doi: 10.3390/ijms23126826.

Single-Step Fast Tissue Clearing of Thick Mouse Brain Tissue for Multi-Dimensional High-Resolution Imaging

Youngjae Ryu  1   2 , Yoonju Kim  1 , Hye Ryeong Lim  1 , Hyung-Joon Kim  3 , Byong Seo Park  4 , Jae Geun Kim  4 , Sang-Joon Park  2 , Chang Man Ha  1
Affiliations

Single-Step Fast Tissue Clearing of Thick Mouse Brain Tissue for Multi-Dimensional High-Resolution Imaging

Youngjae Ryu et al. Int J Mol Sci. .

Abstract

Recent advances in optical clearing techniques have dramatically improved deep tissue imaging by reducing the obscuring effects of light scattering and absorption. However, these optical clearing methods require specialized equipment or a lengthy undertaking with complex protocols that can lead to sample volume changes and distortion. In addition, the imaging of cleared tissues has limitations, such as fluorescence bleaching, harmful and foul-smelling solutions, and the difficulty of handling samples in high-viscosity refractive index (RI) matching solutions. To address the various limitations of thick tissue imaging, we developed an Aqueous high refractive Index matching and tissue Clearing solution for Imaging (termed AICI) with a one-step tissue clearing protocol that was easily made at a reasonable price in our own laboratory without any equipment. AICI can rapidly clear a 1 mm thick brain slice within 90 min with simultaneous RI matching, low viscosity, and a high refractive index (RI = 1.466), allowing the imaging of the sample without additional processing. We compared AICI with commercially available RI matching solutions, including optical clear agents (OCAs), for tissue clearing. The viscosity of AICI is closer to that of water compared with other RI matching solutions, and there was a less than 2.3% expansion in the tissue linear morphology during 24 h exposure to AICI. Moreover, AICI remained fluid over 30 days of air exposure, and the EGFP fluorescence signal was only reduced to ~65% after 10 days. AICI showed a limited clearing of brain tissue >3 mm thick. However, fine neuronal structures, such as dendritic spines and axonal boutons, could still be imaged in thick brain slices treated with AICI. Therefore, AICI is useful not only for the three-dimensional (3D) high-resolution identification of neuronal structures, but also for the examination of multiple structural imaging by neuronal distribution, projection, and gene expression in deep brain tissue. AICI is applicable beyond the imaging of fluorescent antibodies and dyes, and can clear a variety of tissue types, making it broadly useful to researchers for optical imaging applications.

Keywords: deep brain tissue; light sheet fluorescence microscopy; low viscosity; molecular imaging; refractive index matching solution; simple immersion; tissue clearing.

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Conflict of interest statement

These authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Technical limitations in multidimensional imaging and development of AICI solution. (A) Air bubbles on the sample surface or inside tissue caused aberrations in imaging using light microscopy. Multidimensional images by fast confocal microscopy (upper panel) and Lightsheet Z.1 (lower panel) show that bubbles impede the 2D and 3D rendering of images in cleared brain tissue by CLARITY. Red arrows represent bubble obstacles. Scale bars, 200 µm. (B) Comparison of tissue transparency and distortion by reagent change. The 1 mm-thick brain slice was treated with different components of the AICI solution. (a) Brain slice treatment with 10% (wt/vol) N-methyl-D-glucamine, 17% (wt/vol) D-Sorbitol, 22% (vol/vol) TDE, 0.5% (wt/vol) saponin, 1% (vol/vol) Triton X-100, 0.5% (wt/vol) α-thioglycerol, and 0.025% (wt/vol) NaBH4 for 90 min. Refractive index was 1.463. (b) Brain slice treatment with 20% (wt/vol) N-methyl-D-glucamine, 30% (vol/vol) TDE, 45% (wt/vol) Iodixanol, 0.2% (wt/vol) Triton X-100, 0.5% (wt/vol) α-thioglycerol, 0.1% (vol/vol) triethanolamine, and 0.25% NP-40 (vol/vol) for 90 min. Refractive index was 1.472. (c) The c solution substituted 1% (wt/vol) saponin for 0.25% NP-40 (vol/vol) and treated the brain slice for 90 min. Refractive index was 1.468. Right panel of each image indicates the sample area, the red-dotted line represents the border of the initial brain slice at 0 min, and the blue-dotted line represents the border of brain slices treated with a different OCA component of the AICI matching solution after 90 min. Scale bars, 3 mm. (C) Representative photographs showing the coronal section of Thy1-GFP-M mouse brain in each OCA component solution of panel B, respectively. Inset of each image shows high-magnification images. Scale bars, 50 µm.
Figure 2
Figure 2
Comparison of tissue transparency and distortion in AICI with other OCA-based commercial RI matching solutions. (A) Representative images of tissue transparency at different temperatures (25 °C, 35 °C) in AICI with other OCA-based commercial RI matching solutions. The 1 mm thick coronal brain slices from Thy1-EGFP mice were treated with each RI matching solution and imaged at the indicated time points. (B) Transmission curves of each indicated RI matching solution (mean ± SEM, n = 3 measurements). (C) Transmission curves of the 1 mm brain slices treated with each indicated RI matching solution. Transmittance was measured along the cortex part of the 1 mm brain slices after 1 h incubation at 35 °C. Data are presented as the mean ± SEM (n = 3 brain slices). (D) Representative images of tissue distortion at the indicated time points (0, 30, 60 min and 24 h) at 35 °C in each RI matching solution. Scale bar, 3 mm. (E) The red-dotted line represents the border of the initial brain slice at 0 min, and the blue-dotted line represents the border of brain slices treated with each RI matching solution after 24 h. (F) Normalized deformation of brain slices after treatment with each RI matching solution. Sample distortion was represented relative to the initial brain slice outlines before (blue dots) and after immersion for 24 h (red dots) in each RI matching solution. The box-whisker plot indicates the mean ± SEM (n = 4). Statistical significance (* p < 0.05) was assessed by one-way ANOVA with Tukey tests.
Figure 3
Figure 3
Fluid flow characteristics of each RI matching solution. (A) Representative images of the comparison of capillary action after exposure to air for 36 h for each RI matching solution. AICI was observed at the highest (red-dotted) line. (B) The hardening process of each RI matching solution in a 24-well plate at 25 °C for 24 h to 30 days. Top shows each RI matching solution contained in the well plate when the plate was laid flat, and when the well plate was tilted. The flow of RI solution is outlined with dashed red lines. Only the AICI solution was retained in a liquid state, whereas other matching solutions hardened and crystallized after immersion for 30 days. (C) The refractive index changes are time dependent. An amount of 500 µL of each RI matching solution was placed in a 24-well plate with air exposure in a laboratory environment and measured for refractive index using an Abbe refractometer (NAR-1T solid, ATAGO, Tokyo, Japan). The refractive index displaying the dotted graph for each RI matching solution and statistical significance (** p < 0.01, * p < 0.05) was assessed by one-way ANOVA with Duncan’s test (n = 3). (D) The viscosity of each reagent was measured by a rotational rheometer system. Insets are enlarged to distinguish the viscosity data of each reagent following step 12. The viscosity of the AICI reagent is closest to that of water (20 steps). Data plot indicates the mean ± SEM (n = 3).
Figure 4
Figure 4
Comparison of long-term imaging and fluorescence preservation in different RI matching solutions. (A) 4% PFA-fixed HEK293T cells in a 24-well cover glass chamber were imaged for morphological changes over time with 500 μL of each RI matching solution using a Nikon perfect focus system (PFS). The cells on the glass bottom in the red inset box represent enlarged images (first and last frame, right). Scale bars, 50 μm. (B) Z-plot profiles show changes in fluorescence intensity in 600 μm of Thy1-GFP-M mouse brain slice by tissue penetration of each RI matching solution. The fluorescence intensity was normalized to the peak value by each RI solution using a confocal microscope with a 10× objective. AICI rapidly penetrated the brain slice tissues and the fluorescence intensity was highly preserved by AICI, rather than the other reagent-based RI matching solutions. (C) Representative images of fluorescence intensity at a median depth (z = 300 µm, grey-dotted lines in (B)) of hippocampal CA1 and cortical regions treated with each RI matching solution at indicated times. Scale bar, 200 μm. Inset scale bar, 100 μm.
Figure 5
Figure 5
Depth capacity and morphology maintenance of AICI with three-dimensional neuronal imaging. (A–C) The penetration and clearing capacity of AICI by sample thickness was performed for the indicated immersion times. The three-dimensional limitations of optical imaging were identified by reconstructing images from a 1–3 mm depth of AICI-treated Thy1-GFP-M mouse brain slices using Lightsheet Z.1 with a 5× objective lens. Sectional images at different 3D depths are shown with a 500 μm z-step. Scale bars, 1 mm. (D) Comparison images of neuronal structures at representative depths after PBS and AICI clearing using Nikon A1Rsi with 100× Plan Apo Lambda Oil lens (N.A 1.40, WD 0.13 mm). Three-dimensional reconstruction of images of neuronal dendrites in PBS (a) and after AICI clearing for 90 min (e). Section images of neuronal dendrites at the indicated depths in PBS (b–d) and after AICI for 90 min (f–h). Scale bars, 5 μm. (E) Thy1-GFP-M mouse brain slice cleared by AICI reconstructed in 3D in the z-axis (100 μm and 40 μm x-y plane). (F) Image in the blue section in (E) shows axonal boutons and dendritic spines after AICI clearing. Right panel shows an enlarged image of the yellow- and red-dotted boxes in the left panel. Scale bar, 5 μm. Inset scale bar, 1 μm. (G) 3D reconstruction of detailed neuronal synapse morphology by AICI clearing represented by the red box in panel (E). Scale bar, 1 μm.
Figure 6
Figure 6
Application of AICI to three-dimensional and functional imaging. (A) 3D-reconstruction of AgRP Cre-tdTomato neurons (yellow), POMC-EGFP neurons (green), and DAPI (blue) images for the nucleus in the mouse brain hypothalamus region. SO, supraoptic nucleus; PVN, paraventricular nucleus; LHA, lateral hypothalamic area; VMH, ventromedial hypothalamic nucleus; ARC, arcuate hypothalamic nucleus; ME, median eminence; TU, tuberal nucleus; and 3V, third ventricle. (B) Representative 3D volume image from the rostral to caudal ARC region by AICI immersion. Each two-dimensional selected single image was sorted from the 3D-reconstructed image (left panel). Scale bars, 100 μm. (C) The 3D reconstructed image of the SO region clearly shows the AgRP cells axially located on the ARC as well as the periventricular hypothalamic nucleus (Pe) of the rostral hypothalamus, whereas the cell bodies of the POMC neurons are predominantly located in the ARC and the retrochiasmatic area (RCh). (D) The 3D expression pattern of AgRP (green), POMC (gray), and c-Fos (red) in the hypothalamic ARC region under fed and fasted conditions. Scale bars, 100 μm. (E,F) The maximum intensity projection images of AgRP, POMC neuronal population, and neurite density in the ARC and VMH regions relatively. Scale bars, 100 μm. (G) Bar graphs displaying quantitative analysis of the integrated density of the AgRP and POMC neuronal cell bodies in each experimental group (n = 4). (H) The 3D-rendered images of AgRP and POMC in the fed and fasted mice easily show the neuronal population, colocalization, and density of neurites. The c-Fos (red) was stained with a primary antibody followed by Alexa Fluor 647 secondary antibody using electrophoretic antibody staining method, as discussed in the materials and methods. The arrow indicates colocalized cell bodies in inset. (I) Bar graph displaying the number of AgRP neurons positive for c-Fos in the ARC in each experimental condition (n = 4 for fed and fasted group, respectively). Note the dramatic population changes and neuronal activation of AgRP/POMC neurons in the fasted ARC region, rapidly and easily identified by only simple immersion in AICI. * p < 0.05, ** p < 0.1.
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