Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Apr;47(3):441-453.
doi: 10.1111/nan.12673. Epub 2020 Dec 2.

Optimised tissue clearing minimises distortion and destruction during tissue delipidation

Krit Lee  1 , Hei Ming Lai  2   3 , Maja Hoejvang Soerensen  1 , Edward Sai-Kam Hui  4 , Victor Wan-San Ma  5 , William Chi-Shing Cho  5 , Yuen-Shan Ho  6 , Raymond Chuen-Chung Chang  1   7
Affiliations

Optimised tissue clearing minimises distortion and destruction during tissue delipidation

Krit Lee et al. Neuropathol Appl Neurobiol. 2021 Apr.

Abstract

Aims: A variety of tissue clearing techniques have been developed to render intact tissue transparent. For thicker samples, additional partial tissue delipidation is required before immersion into the final refractive index (RI)-matching solution, which alone is often inadequate to achieve full tissue transparency. However, it is difficult to determine a sufficient degree of tissue delipidation, excess of which can result in tissue distortion and protein loss. Here, we aim to develop a clearing strategy that allows better monitoring and more precise determination of delipidation progress.

Methods: We combined the detergent sodium dodecyl sulphate (SDS) with OPTIClear, a RI-matching solution, to form a strategy termed Accurate delipidation with Optimal Clearing (Accu-OptiClearing). Accu-OptiClearing allows for a better preview of the final tissue transparency achieved when immersed in OPTIClear alone just before imaging. We assessed for the changes in clearing rate, protein loss, degree of tissue distortion, and preservation of antigens.

Results: Partial delipidation using Accu-OptiClearing accelerated tissue clearing and better preserved tissue structure and antigens than delipidation with SDS alone. Despite achieving similar transparency in the final OPTIClear solution, more lipids were retained in samples cleared with Accu-OptiClearing compared to SDS.

Conclusions: Combining the RI-matching solution OPTIClear with detergents, Accu-OptiClearing, can avoid excessive delipidation, leading to accelerated tissue clearing, less tissue damage and better preserved antigens.

Keywords: antigenicity; delipidation; opacity; protein loss; three-dimensional imaging; transparent brain.

PubMed Disclaimer

Conflict of interest statement

None of the authors have a conflict of interest.

Figures

Figure 1
Figure 1
Tissue clearing with different concentrations of SDS vs SDS‐OPTIClear. (A) Representative images of 4 mm‐thick rat brain tissues before and after clearing with SDS and SDS‐OPTIClear respectively. Scale bar = 5 mm. (B) Absorbance at 750 nm for tissues cleared at varying concentrations of SDS vs SDS‐OPTIClear (n = 5–6). Lower absorbance level indicates higher transparency level. (C) Fold of area change after clearing with SDS compared to SDS‐OPTIClear (n = 3). Tissue sizes were quantified and normalised to the size of tissue before clearing. (D) Fold of protein loss after clearing for SDS only compared to SDS‐OPTIClear (n = 5–6). Solutions used for clearing were collected and then added to a stain‐free gel for electrophoresis, band intensities were quantified and normalised with the protein band intensity of sample solution collected at day 0 (before clearing). Mean ± SEM for all graphs. Two‐way ANOVA, Tukey's multiple comparison test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001
Figure 2
Figure 2
Tissue distortion level after clearing with different conditions. 1 mm‐thick rat tissues were stained with TO‐PRO®‐3 and then imaged before delipidation, after delipidation and after RI‐matching in OPTIClear. (A) Representative images of TO‐PRO®‐3 stained tissues under different stages of clearing. White dotted lines marked the original boundary of tissues. Scale bars = 1 mm (n = 3). (B) Fold tissue area changes after tissue clearing and RI‐matching, respectively. (C) Percentage changes in dorsal–ventral (D‐V) and mediolateral (M‐L) lengths after RI‐matching respectively (n = 3). (D) Percentage changes in the ratio of D‐V and M‐L length after RI‐matching (n = 3). (E) Merged images of GFP‐labelled hippocampal neurons under different stages of clearing. The same neuron was imaged before and after clearing, and after RI‐matching. Images were labelled with different colours (see Fig. S3 and merged together. Higher degree of overlapping indicates lower distortion level in morphology. Scale bars = 20 µm. Mean ±S.E.M. for all graphs. Two‐way ANOVA, Tukey's multiple comparison test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001
Figure 3
Figure 3
Comparison between 3D immunostaining of tissue cleared with SDS and SDS‐OPTIClear. (A) Iba‐1 staining of tissues cleared with 4% SDS‐OPTIClear (left panel) and 4% SDS only (right panel). Upper panel: images taken with a 10× objective. Lower panel: zoomed‐in image of the dentate gyrus (DG) taken with a 20× objective (left image), Z‐stack images taken with a 63× objective with z‐depth = 161.7 μm (right image). (B) TOMM‐20 staining of regions cleared with 4% SDS‐OPTIClear (left panel) and 4% SDS only (right panel). Left image: 20× images of DG regions. Right image: z‐stack of the images showed on the left (z‐depth = 125.84 μm). (C) ChAT staining taken with a 20× objective. Left panel: 20× images of the nbM cleared with 4% SDS and 4% SDS‐OPTIClear. Right panel: z‐stack of the images showed in the left panel (z‐depth = 72 μm). The same imaging and display settings were used for both 4% SDS and 4% SDS with OPTIClear conditions
Figure 4
Figure 4
Comparison of clearing efficiency between combinations of OPTIClear with different detergents. (A) Representative images of tissues before and after clearing (scale bar = 5 mm) (B) Comparison of clearing efficiency between combinations of OPTIClear with SDS and 1,2‐Hexandiol (HxD). Representative images of rat brain hemispheres before and after clearing were shown. (scale bar = 10 mm)
Figure 5
Figure 5
Zebrafish (Danio rerio) imaging after tissue clearing. (A) Whole Tg(fli1a:EGFP) y1 (1.5 years old) after clearing with 4% SDS‐OPTIClear, scale bar = 10 mm. Zoomed‐in 10× image showing EGFP fluorescence in blood vessels. (B)Colour‐coded image labelling the blood vessels in whole zebrafish brain, scale bar = 500 μm. (C) Whole brain immunostained with antibodies against tyrosine hydroxylase, scale bar = 500 μm, higher magnification (20×) showing posterior tuberculum, scale bar = 50 μm
Figure 6
Figure 6
Applications of Accu‐OptiClearing in rodent brain tissues. Visualisation of phosphorylated tau aggregation in 3xTg mice. (A) Image of the hippocampal CA3 region stained with anti‐phosphorylated tau antibody. Inset showing colour‐coded projection of phosphorylated tau‐positive aggregations (indicated by arrowheads in B and C). Scale bar, 50 μm (for A), 10 μm (for B and C). Long‐range tracing of rat nucleus basalis of Meynert neurons with AAV‐GFP. (D) Sagittal rat brain atlas indicating the nbM region in red dotted circle. (E) Tiled Z‐stack image showing AAV‐GFP‐labelled nbM neurons three weeks after injection into the nbM region (scale bar = 1 mm). (F)Colour‐coded projection image with higher magnification of the region shown in E (scale bar = 500 μm, z‐depth = 205 μm)
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Chung K, Wallace J, Kim S‐Y, et al. Structural and molecular interrogation of intact biological systems. Nature. 2013; 497(7449): 332–337 - PMC - PubMed
    1. Yang B, Treweek JB, Kulkarni RP, et al. Single‐cell phenotyping within transparent intact tissue through whole‐body clearing. Cell. 2014; 158(4): 945–958 - PMC - PubMed
    1. Renier N, Wu Z, Simon DJ, Yang J, Ariel P, Tessier‐Lavigne M. IDISCO: A simple, rapid method to immunolabel large tissue samples for volume imaging. Cell. 2014; 159(4): 896–910 - PubMed
    1. Pan C, Cai R, Quacquarelli FP, et al. Shrinkage‐mediated imaging of entire organs and organisms using uDISCO. Nat Methods. 2016; 13(10): 859–867 - PubMed
    1. Murakami TC, Mano T, Saikawa S, et al. A three‐dimensional single‐cell‐resolution whole‐brain atlas using CUBIC‐X expansion microscopy and tissue clearing. Nat Neurosci. 2018; 21(4): 625–637 - PubMed

Publication types

Substances

LinkOut - more resources