Spring 2022 l No.

189 TCIMAIL

Research Article

Basics and Applications of Tissue Clearing Technology

Etsuo A. Susaki
Department of Biochemistry and Systems Biomedicine, Juntendo University Graduate School of Medicine
Laboratory for Synthetic Biology,
RIKEN Center for Biosystems Dynamics Research

Abstract
The recent growth of tissue clearing technology supports the dissemination and generalization of three-dimensional
(3D) observations of complex biological tissues. Since many clearing protocols have been proposed, understanding
the basic principles of tissue clearing is essential for understanding their technical characteristics and selecting the
appropriate one. Practical 3D tissue staining protocols that can stain whole large tissue samples have also been reported.
In addition to these clearing and staining reagents, resources such as light-sheet microscopy and software required for
3D imaging and data analysis have been commercialized and open sourced in recent years. These trends suggest that this
technology will become a more standard basic technology in biomedical research, clinical medicine, and drug discovery
fields.

Keywords: Tissue clearing, light-sheet fluorescent microscopy, three-dimensional imaging, image analysis, three-
dimensional tissue staining

Introduction
Due to its complex three-dimensional (3D) histology is performed by cutting and preparing
structure, biological tissue should primarily be analyzed many sliced sections. The tissue clearing technique is
by 3D information. The optical microscope has a histological method that has shown rapid technological
a desirable resolution that allows cells and subcellular maturity in recent years. Tissue clearing renders light
structures to be resolved in the tissue. However, due to pass through the target tissue and thus allows 3D
to the opacity of the biological tissue, traditional observation of the entire sample without physical
histology does not allow direct observation of internal slicing. This review presents basic knowledge of recent
structures using the optical microscope. Therefore, tissue clearing techniques and their representative
microscopic tissue observation in conventional applications.

Mechanism and procedure of tissue clearing

Tissue clearing is both an old and new technology. one. 3) This study was followed by Tuchin’s physical
The earliest tissue clearing applications by Lundvall investigation in the 1990s and Chiang’s insect tissue
in Sweden and Spalteholz in Germany in the early clearing reagents in the early 2000s. Finally, in 2007,
1900s were an attempt to visually observe the 3D Dodt published whole-organ/body 3D imaging by
construction of tissues. 1,2) More than half a century combining BABB-cleared tissue with a light-sheet
after their early trials, Dent reported a 3D observation microscope (discussed below). 4) This epoch-making
case of frog embryos using Murray’s clear (a mixture work triggered the development of subsequent tissue
of benzyl alcohol and benzyl benzoate, called BABB), clearing technology.
an improved clearing reagent over Spalteholz’s

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Modern tissue clearing technologies include three changes in fibrous proteins are also assumed. 11)
categories as follows:5,6) Therefore, the principle of “RI matching” has not been
(1) a method using organic solvents (hydrophobic completely elucidated.
reagents),
(2) a m e t h o d u s i n g w a t e r - s o l u b l e c o m p o u n d s Organic solvents such as BABB and dibenzyl
(hydrophilic reagents), and ether, and various water-soluble chemicals such as
(3) a method combined with a robust fixation method sugars, alcohols, and aromatic amides can achieve RI
using artificial gels (hydrogel tissue chemistry). matching. Occasionally, RI matching is combined with
BABB and DISCO reagents are representative of additional steps for achieving higher final transparency.
group (1). While these reagents achieve high clearing Delipidation is the step to remove lipids that have
efficiency, issues such as safety and relatively low a high RI and form light-scattering structures such as
signal preservation of fluorescent proteins should be vesicles. Decalcification removes bone hydroxyapatite.
considered. Group (2) includes variations of protocols Organic solvents such as alcohols and tetrahydrofuran,
according to the experimental purposes. Our CUBIC nonionic surfactants such as Triton X-100, and ionic
reagents fall into this category. Group (3) includes surfactants such as SDS and CHAPS are used for
protocols represented by CLARITY, which impart delipidation. EDTA, a representative chelating agent, is
resistance to physicochemically severe conditions such effective for decalcification.
as high temperature and strong tension to biological
tissues. This operation improves the preservation of the Step (2), so-called decolorization and depigmentation,
molecules and allows multiplex labeling. These three aims to remove tissue pigments such as heme and
groups were a convenient categorization method based melanin that absorb light. Removal of heme is
on the background of tissue clearing development. crucial for efficient clearing because the compound
However, several recent integrated protocols such as is particularly abundant in the body and can absorb
PEGASOS and SHANEL combine the advantages visible light shorter than the red wavelength range.
of each group. Therefore, a recent review proposed Decolorization methods had been limited to harsh
a way to understand and classify tissue clearing more treatments such as bleaching. However, we found that
comprehensively.7) amino alcohols enable efficient heme removal under
mild conditions without protein denaturation.12) Melanin
Ti s s u e c l e a r i n g c a n b e a c h i e v e d b y ( 1 ) t h e is a complex biopolymer synthesized from tyrosine.
reduction of light scattering inside the tissue, and (2) So far, there is no efficient way to remove this pigment
the reduction of light absorption inside the tissue. gently, like amino alcohols for heme. Therefore,
bleaching with hydrogen peroxide is usually adopted.
Step (1) is the process for the tissue to be infiltrated Depigmentation of melanin is essential for observing
into a reagent having similar optical properties as pigmented tissues such as in the eyes. 13) However, it
the biological materials (mainly proteins), which should be noted that bleaching eliminates fluorescent
suppresses light scattering and diffraction inside the protein signals.
tissue. Because the refractive index (RI) is considered
a representative index for the optical properties Figure 1 shows a general tissue clearing process
considered here, this step is called refractive index incorporating the mechanisms of steps (1) and
matching (or RI matching). Several protocols aim to (2). Fixed tissue is first treated with delipidation/
lower the macroscopic RI of tissue by swelling. 8–10) decolorization/decalcification reagents, followed by
However, more complicated physicochemical processes immersion into an RI matching reagent, which finally
such as suppression of light scattering due to structural renders the sample transparent.

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 Spring 2022 l No. 189 TCIMAIL

Figure 1. A general procedure of tissue clearing. Part of the figure was created with BioRender.com

We screened the tissue clearing ability of more the whole body of the animal. CUBIC-B for bone
than 1600 water-soluble compounds and invented decalcification is composed of EDTA and imidazole.
a series of CUBIC tissue clearing reagents.14,15) CUBIC-L These CUBIC reagents can clear almost all rodent
has strong delipidation and decolorization abilities organs. For the delipidation of primate specimens,
due to an amino alcohol, N-butyldiethanolamine, including human tissues, CUBIC-HL containing
and a non-ionic detergent, Triton ® X-100. CUBIC-R 1,3-bis(aminomethyl)cyclohexane and a 1,2-hexanediol
is a highly efficient RI matching reagent composed solution 16) is additionally provided. Tokyo Chemical
of two types of aromatic amines. CUBIC-P contains Industry has commercialized these CUBIC reagents.
1-methylimidazole, which has strong decolorizing End users can quickly deploy the quality-guaranteed
activity, in addition to the composition of CUBIC-L. CUBIC products in their experiments.
Perfusion of this reagent allows decolorization from

Three-dimensional imaging and image analysis of cleared tissue

3D observation of cleared samples can be it possible to collect the z-stack of 2D images by
performed with prevalent confocal and multiphoton moving the generated optical sections. Therefore, light-
microscopes. However, light-sheet microscopy is sheet microscopy achieves high-speed 3D imaging. On
further used for faster 3D imaging of large cleared the other hand, this device presupposes a transparent
samples (Figure 2). Light-sheet microscopy creates subject. Therefore, light-sheet microscopy could not
an optical section in a sample with excitation light be applied without achieving advanced tissue clearing
spread out in a sheet shape. This configuration makes technology.

Figure 2. Microscopy for 3D imaging

While prevalent line-scan microscopes can be used for observing a cleared sample, there are issues such as signal leakage from outside the sectioning
plane and long imaging time due to the line-scan operation. Since light-sheet microscopy excites only the acquiring cross-section with sheet-shaped
illumination light, it enables high-speed 3D observation, avoiding the issues of line-scan microscopes.

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Recently, several companies such as Miltenyi 2D staining. The insufficient penetration even of small
Biotec (LaVision Ultramicroscopy), 3i (Cleared molecules suggests that this is due to the complex
Tissue LightSheet), and Bruker (LCS SPIM) have physicochemical environment in the tissue rather than
commercialized light-sheet microscopy for cleared simple reasons such as molecular size.
tissues. In addition, a group at the University of Zurich
has released mesoSPIM, an open-source light-sheet We attempted to solve this problem. We elucidated
system for cleared tissues17) (https://mesospim.org/). In that the tissue has the quite similar physicochemical
the future, cheaper light-sheet systems that non-expert properties as an electrolyte gel. This finding enabled
end-users can easily handle will further contribute to screening of superior 3D staining conditions by
the spread of tissue clearing technology. combining a tissue-mimicking artificial gel and
computer simulation. We finally succeeded in the
The experimenter needs to visualize the cell or bottom-up design of the ideal 3D staining protocol
tissue structure of interest by appropriate labeling. “CUBIC-HistoVIsion (CUBIC-HV ® )” by combining
Genetic tools such as transgenic and knock-in the screened essential conditions. CUBIC-HV can
animals, or viral vectors, are representative labeling uniformly stain ~1 cm 3 3D tissue specimens, such as
methods. Histological staining with small dyes a whole mouse brain and a human brain block, with
and antibodies is also an option. However, in such various antibodies and stains18) (Figure 3). CUBICStars
3D tissue staining, the staining probe requires Inc. has commercialized the CUBIC-HV kits and
penetrating the tissue until it binds to the target. It is sells them via Tokyo Chemical Industry (https://www.
generally difficult to obtain uniform staining inside cubicstars.com/cubic-hv/index.html).
the tissue with a simple extension of the conventional

Figure 3. Development and applications of CUBIC-HistoVIsion

We screened the essential factors for improving the penetration of the staining probes to achieve uniform 3D staining. We clarified two crucial factors:
(1) modulating the interaction between the staining probe and the tissue, and (2) setting appropriate parameters such as the staining temperature (room
temperature or higher) and the initial concentration of the staining probes (5-20 µg/mL antibodies). We finally designed a bottom-up 3D staining and
imaging protocol, “CUBIC-HistoVIsion (CUBIC-HV),” by integrating these results. CUBIC-HV made it possible to uniformly stain and visualize sizable
(~1 cm3) 3D samples, such as whole mouse brain. Figure images were adopted from reference 18 (CC-BY-4.0).

Applying an appropriate quantitative analysis resolution atlas consisting of all cell coordinates in the
is essential for extracting biological information mouse brain, 8) and “CUBIC-Cloud ® ”, a cloud-based
from the acquired 3D image data. As an analysis software for whole-brain analysis and data sharing. 19)
platform for organ-scale 3D image data, we have CUBICStars Inc. provides the commercialized CUBIC-
developed “CUBIC-Atlas,” a whole-brain single-cell Cloud software (https://cubic-cloud.com/).

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Applications of tissue clearing in biomedical research

The modern tissue clearing technique was initially is a representative area because information on 3D
considered for use in neuroscience. Many studies tissue structure is significant. Recent examples include
reported applications in structural and functional analysis of systemic cancer metastasis,25) detection of
analysis of the global neural circuit, such as dopamine micrometastasis, 26) 3D pathology of prostate cancer
circuits, 20) circuits in the frontal cortex, 21) a novel biopsy, 27) and cancer tissue diversity analysis. 28)
taste circuit, 22) and whole-brain circuits responding The technique has also been used in the clearing of
to drugs. 23,24) However, researchers in various fields spheroids and organoids.29) Some recent protocols can
have also adopted this technique. Cancer research even make crustaceans and insects transparent.30,31)

Conclusions
Tissue clearing technology has rapidly spread in areas. Readers who need more detailed information
a wide range of biomedical research and is becoming are encouraged to look over excellent reviews on the
one of the general experimental techniques in several detailed principles, workflows, and applications.5–7,11)

COI disclosure
The author is an employee of CUBICStars, Inc., covering the CUBIC and CUBIC-HV reagents,
and is a co-inventor on patents and patent applications respectively.
owned or filed by RIKEN and CUBICStars, Inc.,
Triton® is a registered trademark of The Dow Chemical Company. CUBIC-HV® and CUBIC-Cloud® are registered trademarks of CUBICStars, Inc.

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Author Information

Etsuo A. Susaki

Dr. Etsuo A. Susaki graduated from Kyushu University School of Medicine in 2002 and
received his Ph.D. (Medicine) from the same university in 2007. His main research interests
are functional states of multicellular systems and their regulatory mechanisms. In particular,
he developed the CUBIC framework and realized a cell-omics approach to study multicellular
systems. Recently, he has successfully developed CUBIC-HistoVIsion, an efficient 3D staining
and imaging technique for organs and the whole body. He is also an expert in molecular
biology, biochemistry, and genetics. He is currently using his unique research background and
techniques to uncover the hidden states of multicellular systems (e.g., brain, organoid) and early
processes of aging/aging-related diseases.

Related Products
Tissue-Clearing Reagent CUBIC-L [for delipidation and decoloring] 25mL 100mL 500mL T3740
Tissue-Clearing Reagent CUBIC-R+(N) [for RI matching] 25mL 100mL 500mL T3983
Tissue-Clearing Reagent CUBIC-R+(M) [for RI matching] 25mL 100mL T3741
Tissue-Clearing Reagent CUBIC-B [for decalcification] 25mL 100mL T3780
Tissue-Clearing Reagent CUBIC-HL [for highly fatty tissue and quenching autofluorescence] 25mL 100mL T3781
Tissue-Clearing Reagent CUBIC-P [efficiently aids perfusion fixation] 25mL 100mL T3782
Tissue-Clearing Reagent CUBIC-X1 [for tissue expansion] 25mL 100mL T3866
Tissue-Clearing Reagent CUBIC-X2 [for RI matching while keeping the expanded size] 25mL 100mL T3867
Mounting Solution (RI 1.520) [for CUBIC-R+] 50mL M3294
Mounting Solution (RI 1.467) [for CUBIC-X2] 50mL M3292
CUBIC-HV™1 3D nuclear staining kit 1KIT C3709
CUBIC-HV™1 3D immunostaining kit (Casein separately) 1KIT C3717

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