Tissue Clearing Techniques 

In the last few years there has been a significant increase in the number of researchers that want to incorporate tissue clearing and 3D histology into their bio-imaging workflows. While there are numerous tissue clearing techniques and versions of these techniques, adopting tissue clearing has been challenging for many researchers as it requires an expertise in tissue clearing and 3D imaging - we are working to make this process easier. 

Which tissue clearing technique is best for me? 

This question unfortunately does not have an easy answer and will depend entirely upon your specific research question. Each tissue clearing technique has advantages and no technique is best for all applications. However, you can narrow down the best clearing technique for your application by considering four different tissue clearing properties using the table below: 

1) Molecular Labeling technique

Tissue clearing techniques are compatible with either only immunolabeling or fluorescent protein (FP) or both. Each approach to tissue clearing has advantages and disadvantages whereas approaches that are compatible with both immunolabeling and FP such as FocusClear™, Sucrose, ClearT2, TDE, CUBIC, CLARITY have their own specific disadvantages such as cost, tissue shrinkage, toxicity, slow clearing, tissue swelling and complexity, respectively. While techniques that are compatible with immunolabeling can be used with FP through using anti-FP immunolabels, this might increase overall processing time when compared to techniques that are compatible with FP. 

2) Processing Time

Overall tissue processing and clearing time is a major driver for which technique should be chosen for an application as this will directly impact the rate at which the technique can be adopted. The time required to render a tissue transparent will depend on several factors including; tissue type, temperature, tissue size, animal age and tissue preparation method (e.g. fixed or fresh). Additionally, each clearing technique will react differently to each one of these parameters whereas solvent based techniques can easily render fatty tissues transparent and lipid removal techniques like CLARITY cannot. 

3) Ease-of-use

The use of protein expansion/denaturation clearing techniques (e.g. Scale) and solvent based techniques (e.g. Visikol HISTO, i/3/uDISCO, BABB) are relatively simple whereas tissues are placed in varying solutions until the tissues are rendered transparent and are able to be visualized on the microscope. These processes lend themselves for high-throughput applications and can be successfully executed by any laboratory technician. Conversely, hydrogel-based approaches (e.g. CLARITY) require embedding tissues in acrylamide hydrogels which is a significantly more challenging process. Though companies (Logos Biosystem, LifeCanvas Technologies) have developed CLARITY devices to automate such processing, these techniques require expensive equipment (>$15,000) and are challenging. 

4) Validation

Most researchers who are using tissue clearing for basic research do not need to be concerned with validation whereas researchers and clinicians using tissue clearing for applied research or on clinical tissues do need to consider validation. For some applications researchers need to be certain that the 3D renderings acquired from tissues are indicative of the tissue’s molecular and morphological properties and not the tissue clearing process. Therefore, these 3D renderings need to be correlated to the traditional histological methods that have been developed over the last century that serve as the foundation for tissue characterization. The challenge in validating these approaches is that while they can be used to acquire 3D renderings of tissues, many of the processes tend to significantly alter cellular morphology, leading to the expansion or shrinkage of tissue. Additionally, some clearing processes (e.g. CLARITY) require the removal of cellular components (e.g. lipids) from tissues to render tissues transparent. Combined, these factors lead to irreversible damage to the cellular morphology of tissues which necessitates parallel validation for these tissue clearing approaches. Therefore, the need for validation is a significant consideration for some researchers whereas the need to validate 3D histology requires parallel 2D histological processing or a tissue clearing technique that is non-destructive and reversible. It has been shown that both iDISCO and Visikol HISTO are reversible as opacity can be returned to tissues following imaging such that 2D histology can be conducted after 3D imaging with the same tissue [1].

Tissue Clearing Table 

Class Name RI Immunostaining FP Clearing Time* Morphology Alterations Preservation of Lipids Toxic / Teratogenic Cost ($US /mL)
Solvent clearing techniques BABB 1.56 Yes No Days Shrinkage No Yes $0.30
FluoBABB 1.56 No Yes Days-Weeks Shrinkage No Yes $0.45
3DISCO 1.56 Limited Quenches Hours-days Shrinkage No Yes $0.20
iDISCO 1.56 Yes Quenches Hours-days Shrinkage No No $0.20
uDISCO 1.58 Yes Quenches Hours-days Shrinkage No Yes $1.07
Visikol HISTO 1.51 Yes Quenches Hours-days No Yes No $1.20
Aqueous hyper-hydrating clearing techniques Sucrose 1.44 Yes Yes Days Shrinkage No No $0.05
FocusClear 1.47 Yes Yes Hours-days No Yes Yes $78.00
ClearT 1.44 Yes No Hours-days No Yes Yes $0.98
ClearT2 1.44 Yes Yes Hours-days No Yes Yes $0.42
SeeDB 1.48 No Yes Days No Yes No $0.29
FRUIT 1.48 No Yes Days Minimal expansion Yes No $0.88
TDE 1.42 Yes Yes Days-weeks No No No $1.04
ScaleA2 1.38 Limited Yes Weeks-months Expansion No No $0.50
ScaleS 1.47 Yes Yes Days-Weeks Expansion, then restored No No $0.45
CUBIC 1.47 Yes Yes Days-Weeks Expansion No No $1.10
ScaleCUBIC 1.47 No Yes Days-Weeks Expansion, tissue becomes very fragile No No $1.44
Hydrogel embedding techniques CLARITY 1.45 Yes Yes Days-Weeks Expansion No Yes (acrylamide monomer)
PACT 1.38 to 1.48 Yes Yes Days-Weeks Slight expansion No Yes
PARS 1.38 to 1.49 Yes Yes Days No No Yes

I have cleared and labeled tissue - now what? 

The second part of the 3D histology process is to image tissues that have been rendered transparent with a confocal, light sheet or two-photon microscope. These imaging modalities also have their own advantages and disadvantages whereas the modality you should use is based upon your specific research question. For example, light sheet microscopy is very good at acquiring 3D images from large whole tissues while it is limited in super high resolution imaging compared to confocal microscopy. There are two very important considerations for imaging that will help guide you to the right imaging modality. 

1) You cannot see greater than 500 to 1,000 microns with air-matched objective on a confocal instrument

Commonly, researchers will encounter a problem in which it appears that their labels have not penetrated their tissues. However, the majority of the time this is due to a misunderstanding about the limitations of ones instrument. Because of differences in refractive index between objectives and solutions, traditional air objectives are limited to approx. 500 to 1,000 microns in imaging depth even if they have very long working distances. To see deeper, a researcher can section their tissue into multiple 1-2 mm sections, purchase an RI-matched dipping objective (>$10,000) or use a light sheet microscope. 

2) Will your tissue clearing solution (e.g. Visikol HISTO, BABB, i/3DISCO) destroy your dipping objective

The short answer is that you cannot be sure unless you ask your manufacturer, but just don't try it with your expensive objective. Most objectives are currently designed for water, glycerol or oil and should not be placed in a solvent (Visikol HISTO, BABB, i/3DISCO). To use a solvent based clearing technique with your objective you can use a double chambered cuvette wherein the outer chamber is filled with the medium matching your lens (e.g. glycerol) and the inner chamber with Visikol HISTO. For light sheet microscopes, the sample can be placed in a cuvette within the imaging cuvette or you can use the Ultramicroscope that is compatible with all solvents.  

Imaging Core Map


[1] Royen, M. E., Verhoef, E. I., Kweldam, C. F., Cappellen, W. A., Kremers, G. J., Houtsmuller, A. B., & Leenders, G. J. (2016). Three‐dimensional microscopic analysis of clinical prostate specimens. Histopathology69(6), 985-992.

[2] Richardson, D. S., & Lichtman, J. W. (2015). Clarifying tissue clearing. Cell, 162(2), 246-257.

[3] Berke, I. M., Miola, J. P., David, M. A., Smith, M. K., & Price, C. (2016). Seeing through musculoskeletal tissues: improving in situ imaging of bone and the lacunar canalicular system through optical clearing. PloS one, 11(3), e0150268.

[4] Pan, C., Cai, R., Quacquarelli, F. P., Ghasemigharagoz, A., Lourbopoulos, A., Matryba, P., ... & Ertürk, A. (2016). Shrinkage-mediated imaging of entire organs and organisms using uDISCO. Nature Methods.