INTRODUCTION
Biological tissue is a three-dimensional (3D) structure with complex detailed structures and vast molecular information, and how to acquire these data has always been a critical challenge [14, 20, 28, 35, 37, 43, 44, 54]. This task has mainly relied on cutting the tissue into thin slices and providing cellular information at the 3D level. Such conventional tissue imaging inevitably loses crucial molecular information after tissue slicing and also artificially segment interconnection and function of tissues, which is particularly important for neurological studies. Therefore, how to transform intact tissues and organs into optically transparent structures has attracted people’s attention. Tissue optical clearing, a unique large volume imaging method, enable tissue structures to be visualized in the whole tissue or in the whole body that has not been sliced. Tissue clearing methods achieve deeper imaging in large volumes by reducing scattering and increasing the depth of light penetration [29, 45–47, 53]. Furthermore, these methods can adapt to various optical imaging techniques and show great potential to acquire 3D high-resolution images of intact tissue [5, 32].
At present, there are mainly three types of tissue clearing methods: hydrophobic, hydrophilic and hydrogel methods [39, 41]. Hydrophobic tissue clearing methods use organic solvents to achieve rapid transparency of intact tissue by reducing light scattering [44]. 3DISCO, a hydrophobic tissue clearing method, can completely clear the intact adult mouse brain within 2 days. However, the storage time of samples is short owing to fluorescence quenching [10, 11]. The hydrophilic tissue clearing methods are carried out by using water-soluble reagents. Although the tissue clearing ability of hydrophilic tissue clearing methods (such as Scale, Scales, SeeDB, CUBIC, etc.) was sometimes inferior to that of hydrophobic tissue clearing methods, they offer unique advantages, including preservation of protein function and high levels of biosafety and biocompatibility [16, 17, 19, 38]. The hydrogel-based tissue clearing method is similar to the hydrophilic tissue clearing method, but the lipids are extracted from the hydrogel-tissue hybrid in a more aggressive way.
CLARITY (Clear Lipid-exchanged Acrylamide-hybridised Rigid Imaging/Immunostaining/In situ hybridisation-compatible Tissue-hYdrogel) proposed in 2013 is a hydrogel-based tissue clearing method, and its necessary steps are as follows: tissue fixation, hydrogel formation, lipid extraction, staining, and imaging [3]. By crosslinking acrylamide with intact tissue to form a reticular hydrogel structure, this technique provides a physical framework for tissue without destroying biomolecules and fine structure characteristics of removing lipids from intact tissue to turn it into optically transparent and permeable transparent tissue. Therefore, it can be stained and de-stained for multiple rounds without tissue sections, and then the 3D structure of the tissue can be observed and constructed under an optical microscope. CLARITY was initially applied to construct the structure of brain tissue, showing neurotransmitters, cellular relationships, subcellular structures intact-tissue imaging of long-range projections, local circuit wiring, etc. It has been widely used in the basic research of the pathological structure of nervous system-related diseases, transparent processing of intact organs (such as pancreas, lung, intestine, liver, and kidney), the relationship between organ structure and function and dynamic changes of blood vessels, 3D imaging of plant organs, 3D biogeography and metabolic status of microorganisms and other scientific research [1, 3, 4, 12, 25, 31, 36]. Furthermore, CLARITY can also be combined with traditional imaging techniques, which brings new opportunities for the fundamental study of disease pathology [36].
However, the variety and complexity of the improved techniques for CLARITY, as well as such challenges as complicated steps, low tissue clearing efficiency, tissue damage, and expensive experimental equipment significantly limited popular application. This review systematically summarises the research progress of CLARITY techniques from the perspective of tissue clearing. The brief schematic representation of CLARITY and its modifications is shown in Figure 1.
In addition, since the lipids in the tissue affect the tissue transparency to a great extent, the focus of CLARITY is the extraction of lipids from the tissue [22]. Lipid extraction can be passive thermal diffusion, with lipids captured in the detergent micelle diffusing, or active by applying an electric field. The two different methods of lipid extraction are compared in Table 1.
Methods |
Electrophoresis |
Passive thermal diffusion |
Clearing speed |
Rapid (several hours to days) |
Slow (several days to weeks) |
Sample damage |
Severe |
Mild |
The loss of biological information |
More |
Less |
Cost and complexity |
Expensive and difficult |
Cheap and easy |
Circulation |
Need (to control the flow rate and temperature) |
Needless |
Clearing buffer |
4% SDS (in PBS buffer, pH 8.5) |
8% SDS (in PBS buffer, pH 7.5) |
According to the way of lipid extraction, these modified CLARITY are classified as active CLARITY, passive CLARITY, and the method of merging active CLARITY with passive CLARITY to select the appropriate tissue clearing method more quickly and effectively based on the balance of tissue clearing speed and tissue transparency of different tissue clearing methods. Furthermore, combing through the advantages of CLARITY and highlighting limitations may be conducive to the construction of different research ideas and enlighten to improve the details of the techniques.
PASSIVE CLARITY
The diffusion barrier properties caused by the lipid bilayer of cells are related to chemical penetration and light scattering properties of the lipid-water interface, making the tissue difficult to visualise [3, 40, 54]. Therefore, lipid extraction is critical for the optical transparency of tissue. The original CLARITY used 4% sodium dodecyl sulphate (SDS) (sodium borate buffer, pH 8.5) at 37–50oC for electrophoretic tissue clearing. However, this may be difficult to implement and may lead to variability in the final tissue quality, including epitopes loss, damage to fine structure and tissue damage caused by heating. Fortunately, lipid extraction can be passive: lipids captured in the detergent micelles can slowly diffuse out of the tissue into the detergent solution. Temperature and solution affect the rate of lipid clearing, tissue homogeneity and protein loss, but excessive temperature can also lead to tissue damage and fluorescence quenching. Tissue clearing can also accelerate the removal process by gentle shaking and constantly changing the clearing solution. Poguzhelskaya et al. proposed a simplified diffusion-based method, omitting the electrophoresis chamber, called CLARITY2 [33]. The sample (1–1.5 mm thickness) can be completely cleared by incubating it in the clearing solution for 1–2 weeks, with gentle shaking at 37oC. Passive tissue clearing omits the time-consuming and laborious electrophoresis step and avoids the risk of tissue damage. Moreover, it was reported that there was no significant difference in protein concentration before and after between active and passive clearing [8].
However, several limitations and challenges are facing passive tissue clearing. The main weakness of passive clearing methods is the slow clearing speed, which makes them unsuitable for the clearing of large tissue volumes or whole organisms. Moreover, excessive clearing time leads to a decrease in the signal of fluorescent proteins and an increase in the loss of biomolecules. Therefore, passive CLARITY is an excellent choice for poor laboratory conditions, small samples (tissue thickness < 3 mm), or high requirements for the integrity of tissue structure and molecular information. However, in recent years, how to promote rapid, whole-brain, and whole-body passive clearing has gradually become the focus of research, and some new modified techniques have emerged that have dramatically improved the scope of application of passive CLARITY technique.
PACT and its variants
In the original CLARITY, the experimental animals were transcardially perfused with hydrogel monomer solution (including 4% paraformaldehyde [PFA], 4% acrylamide, and 0.05% bis-acrylamide in phosphate buffered saline [PBS] supplemented with 0.25% VA-044 initiator). The interesting parts were selected and incubated in 4oC hydrogel monomer solution for 3 days and then polymerised into a hydrogel mesh [3]. Although hydrogel-tissue hybrid avoids tissue damage and the loss of biological information, the dense hydrogel mesh structure leads to low SDS transport efficiency and slow down the speed of lipid extraction. Therefore, Yang et al. [52] developed a simple version of CLARITY based on the passive lipids extraction and termed it PACT (passive clarity technique) to increase the efficiency of SDS transportation and retain the fine structure of the tissue. They perfused and fixed tissue with 4% PFA, and then the tissue was incubated in A4P0 (4% acrylamide in PBS supplemented with 0.25% VA-044 initiator) at 4oC overnight. Compared with original CLARITY, the tissue treated with A4P0 increased the hydrogel mesh porosity, which significantly improves the SDS transport speed, but the tissue was also more easily to swell, and the changes of the tissue weight and volume were 174% and 223%, respectively. Fortunately, the tissue will gradually shrink to the original size in the following steps, and this change of tissue volume expansion-contraction has little effect on the tissue structure, even providing an advantage for the application of some dense tissues, such as bone [15]. Moreover, since the hydrogel cross-linking inside and outside the tissue is reduced by removing bis-acrylamide from the hydrogel monomer solution during the hydrogel tissue embedding process, the tissue can simply be removed from the surrounding hydrogel, reducing the physical damage to the sample. Besides, they emphasized that increasing the concentration of the SDS clearing buffer from the original 4% to 8% can achieve uniform tissue clearing, and called 8% SDS buffer as PACT reagents. PACT increases the efficiency of passive lipid extraction and is suitable for clearing small or particularly fragile samples and organs, which can clear 1–3 mm thick samples (including the brain, spinal cord, kidney, heart, lung and intestine, etc.) within 2–5 days.
However, PACT is still slow to clear large volumes of tissues, which limits its scope of application. The PACT reagents can be delivered either intracranially or via the vasculature to clear and label whole-brain and whole-body. A4P0 and PACT reagents were continuously circulated in the fixed animal vasculature by the peristaltic pump for tissue clearing in situ. This method, similar to cardiac perfusion, was named PARS (perfusion-assisted agent release in situ) by Yang et al. [13, 52]. PARS increases the contact area between clearing buffer and tissue, so the rate of tissue clearing becomes faster. Furthermore, the tissue swell of PARS is reduced due to the limitation of physiological structure in the process of tissue clearing. Unfortunately, the implementation complexity of PARS limits its further application. Moreover, PACT/PARS needs more time to get maximal tissue transparency. Therefore, there are some PACT-based tissue clearing methods emerged.
Woo et al. [49, 50] modified PACT by treating tissues in 4% acrylamide and 0.25% VA-044 in two separate steps during hydrogel formation to improve the lipid removal speed, called psPACT (process separation PACT). This separation process avoids the removal of the remaining unpolymerised hydrogel monomers surround the tissue and the time required for lipid extraction of psPACT is also 10% less than that of PACT. Meanwhile, based on the psPACT, Woo et al. [49, 50] added 0.5% a-thioglycerol, a un-browning agent, to the PACT reagent to increase the lipid removal speed of most tissues by 25%, and called this method mPACT (modified PACT). The addition of 0.5% a-thioglycerol to PACT reagent accelerated optical transparency but increased tissue fragility. Therefore, Woo et al. [48] proposed the process separation PACT-Acrylamide (psPACT-A) and modified PACT-Acrylamide (mPACT-A) protocols, which avoid increasing tissue fragility without reducing optical transparency and can be used to remove mouse embryos. In the modified methods, after the tissue was embedded in 0.25% VA-044, the sample was reincubated in the A4P0 solution to improve the firmness of hydrogel. Both psPACT-A and mPACT-A can achieve rapid optical tissue clearing within the same clearing time as the original protocols.
PACT and its variants expanded the application of CLARITY, which can be used to achieve whole-brain and whole-body clearing and labelling. Moreover, these modified methods provide a powerful tool to study the mechanism of disease onset and progression, and can reflect detailed changes in a part of the observed sample as a whole to fully understand its biological characteristics.
Free of acrylamide SDS-based tissue clearing
The original CLARITY includes a hydrogel embedding step based on two hypotheses:
For these reasons, Liu et al. [27] developed an improved CLARITY named FASTClear (Free-of-acrylamide SDS-based Tissue Clearing). 4% PFA/10% neutral-buffered formalin fixed tissue, avoiding the use of hydrogel, is directly immersed in 4% SDS for degreasing and at a temperature of 50°C to increase the removal rate. However, prolonged PFA fixation time will hinder the clearing rate and immune labelling due to excessive PFA cross-linking on the tissue [26]. Moreover, some tissues (such as spinal cord and cortical white matter) are difficult to become transparent after SDS delipidation. Fortunately, iDISCO can permeate optically opaque, PFA-fixed tissue in a cocktail of detergent to immunostain the tissue [34]. Then, the tissue can be optically transparent with organic solvents using the 3DISCO [9]. FASTClear is a simplified and rapid method of tissue clearing, which simplifies the process and adds potential additional benefits, including avoiding the use of toxic acrylamide, increasing antibody penetration, reducing tissue swelling, and increasing compatibility with other tissue treatment techniques. Furthermore, FASTClear can be applied to the imaging of tissues that are difficult to become transparent with traditional tissue clearing techniques, such as fresh spinal cord tissue. However, the FASTClear is limited to antibody labelling, and the penetration depth of the antibody is not uniform, which may be related to the local tissue structure.
Thus, Xu et al. [51] developed a rapid free of acrylamide SDS-based tissue clearing method by modifying and merging PACT and FASTClear to reduce tissue clearing time and preserve fluorescent signals. In the step of lipid extraction, FACT used 8% SDS to remove lipids and removed the process of hydrogel perfusion and embedding from PACT to improve the speed of clearing. The staining step of FASTClear was used for reference, but the working temperature was reduced to 37 oC and the pH 7.5 clearing buffer was used, which could reduce the fluorescence quenching rate. The FACT is a simple and low-cost method of tissue clearing, which improves the preservation of cell structure, the penetration depth of antibody and the long-term retention of the fluorescence signal. Moreover, FACT avoids the use of hydrogel and reduces tissue swelling, which can be applied to the study of fine cytoarchitectural details such as microglia.
ACTIVE CLARITY
Accelerating the extraction of lipids from intact tissue is the key to obtain better tissue imaging. Passive lipid removal has been greatly improved, such as CLARITY2, PACT, PARS, mPACT and other modified methods, which greatly shorten the time of tissue clearing. However, the lipid extraction speed of passive clearing methods is still slow. Therefore, improvements in active clearing methods have been underway. In the original CLARITY, the SDS detergent can capture lipids in SDS micelles, which are negatively charged at alkaline solutions (pH 7.5–8.5), so they can be removed under the action of an electric field in a customized electrophoresis chamber. The electrophoretic tissue clearing (ETC) system of the original CLARITY has four elements [3]:
Electrophoretic tissue clearing increases the lipid extraction speed by several orders of magnitude compared with passive clearing, and make larger samples (such as mouse brain) transparent in a few hours to days, rather than a few days to weeks, significantly increasing the rate of lipid extraction and reducing tissue swelling.
Although ETC enhances the speed of lipid extraction, it also faces some challenges. First, the too strong electric field of ETC will lead to the formation of bubbles, discoloration, and deposition of black particles on the surface of samples, making the hydrogel opaque, and larger bubbles may cause the hydrogel to break. Moreover, the electrophoresis chamber used by the active CLARITY is heated up due to the effect of Joule heating. Although the ETC clears the tissue more thoroughly at higher temperatures, samples tend to lose structural integrity due to high temperature, just like passive CLARITY. In addition, ETC uses a static electrostatic field to cause lipids to be extracted in one direction, resulting in uneven tissue clearing. Furthermore, expensive electrophoresis equipment and complex implementation steps limit the application of electrophoretic tissue clearing. In response to these challenges, there have been many modified methods about ETC in recent years. These methods are summarized in Table 2
Methods |
CLARITY |
Stochastic electrotransport |
Active clarity technique |
PRE-CLARITY (NCES) |
Electrodes |
Wire-electrodes |
Wire-electrodes |
Platinum plate-electrodes |
Wire-electrodes |
Electric field |
Static |
Rotational |
Static |
Static |
Clearing speed* |
++ (2 weeks) |
+++ (3 days) |
++++ (5–6 h) |
+ |
Sample damage |
++ |
+ |
+++ |
++ |
Complexity |
++ |
+++ |
++ |
+ |
Circulation |
Yes |
Yes |
Yes |
No |
and diagrams of these ETC systems are presented in Figure 2.
Stochastic electrotransport
The electric field can drive the movement of charged particles in the tissue with pores (such as hydrogel–tissue hybridisation), but if the tissue contains charged molecules, the electric field can also destroy the tissue. Moreover, the electrical properties in different parts of the tissue are not uniform, and different regions have different electrical properties, which may lead to the emergence of a concentrated electric field in the tissue. Therefore, when lipids are extracted using the static electric field (an electric field that is constant along a certain direction), different parts of the tissue are extracted to different degrees of lipids, which also results in uneven penetration of antibodies in the subsequent steps of staining [21]. The excessive electric field can cause tissue deformation or even tissue structural damage, and may cause bubbles to form inside the hydrogel, reducing the transparency of the tissue and even causing the hydrogel to rupture [3, 21]. Therefore, ETC can accelerate lipid removal in a non-destructive way only at a low electric field, and dense or larger tissue cannot be effectively removed in the low electric field.
Kim et al. [21] proved that the rotational electric field could improve the diffusivity of charged molecules in porous samples, and there is a quadratic correlation between the diffusivity and the electrical properties of charged molecules. Moreover, this electrophoresis-driven diffusion transport method can selectively promote the dispersion of free-moving molecules with high electromobility and inhibit the dispersion of endogenous cross-linked biomolecules with low electromobility in the samples. Therefore, Kim et al. [21] improved the method of transportation called stochastic electrotransport, which does not use the unidirectional field but uses a rotational electric field that changes the direction of the electric field over time. The rotational electric field is achieved by continuously rotating the sample chamber between the two electrodes to generate an external rotational electric field relative to the sample, which enhances the random diffusivity of charged molecules, thereby enhancing the efficiency of lipid extraction and staining of large and dense tissues with nuclear dyes, proteins, and antibodies. Moreover, stochastic electrotransport used nanoporous membranes to contain samples, preventing samples from directly contacting electrodes or electrolytic by-products. Stochastic electrotransport can completely remove the mouse organs within 1–3 days and stain them within 1 day. The advantage of stochastic electrotransport is that it can quickly transport charged molecules out of the tissue without causing significant damage to the tissue structure. Comparing the deformation degree of tissue before and after optical clearing, there is no significant difference in the degree of tissue deformation caused by diffusion and stochastic electrotransport. On the contrary, the extraction of lipids by the static electric field may cause severe damage to the tissue, especially in the tissues with poor conductivity. Although the use of electrophoresis to remove tissue may not use an almost high voltage, the speed of electrophoresis will eventually be limited by tissue damage. Therefore, stochastic electrotransport has apparent advantages in clearing large and dense samples.
However, stochastic electrotransport has the potential for rapid lipid removal and staining, but the temperature increases with the strength of the electric field due to the effect of joule-heating. As mentioned before, the intact tissue structure and biological information will be gradually destroyed and lost as the temperature increases, which limits the further improvement of the clearing efficiency [8]. Therefore, it is significant to explore the balance point between the efficiency of lipid removal, temperature and electric field strength for finding the appropriate temperature and electric field strength of different tissues and organs in the following studies.
Active clarity technique
The use of electrophoresis has dramatically increased the speed of tissue clearing, but the whole process is slow and requires complex procedures. Therefore, Lee et al. [24] improved CLARITY by optimising the composition of hydrogel and electrophoresis and called this method active clarity technique (ACT). As for lipid extraction, there are two specific improvements in the ACT:
Active clarity technique can clear most adult mouse tissues within 2–20 hours, and can expand to large organs (such as the brains of rats or rabbits) and even the whole bodies of adult animals (such as adult mouse, zebrafish and Xenopus) [24]. Comparing the clearing efficiency of ACT with other tissue clearing methods, it has been found that ACT takes 2 hours to achieve almost complete optical transparency of a 1 mm thick brain slice, but it takes 3 days for other methods to achieve similar optical transparency. Therefore, ACT has a great advantage in clearing thick tissues and large organs, even adult animals. The tissue cleared by ACT also showed a high contrast between connective tissue and soft tissue in PBS buffer, and the tissue structure could be distinguished even under a standard anatomical microscope. ACT, a reliable and rapid method for clearing large samples, accelerates the speed of tissue clearing and has promising applications in the field of cellular and molecular functions. However, when ACT is applied to the whole body clearing, the clearing efficiency of different tissues varies with size and density. Moreover, under conventional conditions, ACT clears some tissue with significant swelling, which requires further improvement.
Fix-Clear
The original CLARITY creatively applied hydrogel mesh to bind tissue proteins and nucleic acids to preserve the biological information of the tissue when using detergent to clear lipids. However, subsequent studies have found that hydrogel embedding is not essential for tissue clearing, and some protocols, such as SWITCH, FASTClear and FACT, use SDS for delipidation without hydrogel embedding [27, 30, 51]. Although the formation of hydrogel-tissue complexes provides lasting physical strength in the degreasing process and provides expandability, dry-ability, and the ability to selectively bind to functional chemicals, the process of hydrogel embedding has some drawbacks [7]:
Passive CLARITY first tried the method of SDS-based tissue clearing without acrylamide and made some progress. Therefore, whether this method can be applied to active CLARITY to improve tissue clearing method has attracted attention. Choi et al. [2] merged the acrylamide-free tissue clearing technique with ETC and named it Fix-Clear (FxClear). FxClear has some advantages:
Furthermore, one potential problem with FxClear is that ETC can damage the tissues. Quantitative analysis of protein loss shows that ETC > 1 day can lead to protein loss, which may be caused by tissue damage, so long-term use of ETC is not recommended. On the contrary, only a small amount (< 2%) of protein loss was detected in a safe time (< 1 day). FxClear is particularly suitable for the 3D pathological examination of small samples (such as brain samples of 1~2 mm). Furthermore, FxClear can be applied to some studies that require precise preservation of sample size due to the small tissue swelling.
THE METHOD OF MERGING ACTIVE AND PASSIVE CLARITY
Lipid extraction is essential for obtaining optically transparent tissue. Although electrophoresis is faster than passive thermal perfusion, it needs to establish a professional circulatory system, which makes electrophoresis difficult to manipulate. Therefore, how to design a simplified electrophoresis device to facilitate implementation has become a challenge. Du et al. [6] separates the electrode from the electrophoresis chamber and designs a mobile electrophoresis device named non-circulation electrophoresis system (NCES). Furthermore, Du et al. [6] also reported a new tissue clearing method called PRE-CLARITY (Passive pRe-electrographysics CLARITY) by merging passive thermal perfusion and electrophoretic lipid extraction for the first time. First, the A4P0B0 (4% PFA in PBS) treated brain was cleared by NCES for 1 day with 4% SDS clearing buffer containing 1% alpha-thioglycerol. Then, the passive clearing was conducted in 4% SDS clearing buffer with 5% alpha-thioglycerol at 50°C, with gentle shaking. PRE-CLARITY can make the brain completely transparent in 5 days. They had three main findings:
However, PRE-CLARITY also has some challenges. A4P4B0.05 treated sample was not well cleared in NCES, which may be related to the short electrophoresis time. It may also be related to the flow-assisted dissociation of lipid molecules because the main difference between the original electrophoresis system and the NCES system lies in the flow of clearing buffer. Moreover, NCES uses platinum wire to make electrodes. Platinum plates are reported to generate denser currents, thus speeding up tissue clearing, which can be improved in follow-up research. In short, PRE-CLARITY provides a simplified electrophoresis system and a faster and more transparent method of tissue clearing. The attempt to merge different tissue clearing methods also provides a new perspective for tissue optical clearing method and facilitates the development of tissue clearing method.
CONCLUSIONS
With the deepening of biomolecular information research, CLARITY and its variants will be increasingly applied to fine structure imaging of tissues and the function of cells or biomolecules in intact tissues, which has a broad application prospect. A list of variants of the CLARITY simplify and improve the experimental conditions, equipment, and concrete steps of the original CLARITY. By classifying these modified CLARITY techniques and comparing the similarities and differences between them, it is easier to find the areas where these protocols need to be improved, and we can also learn some improved ideas from them. For example, the hydrogel matrix for passive tissue clearing was optimised to the A4P0 in the PACT protocol, but this formula was later used in active tissue clearing methods. These modified methods increase the speed of tissue clearing and tissue transparency and maintain the fine structure of tissue, which is helpful to the study of different tissue types. Moreover, these improvements also break the limitation of original CLARITY. Optical clearing of intact tissues can be carried out even in ordinary laboratories, making it easier for researchers to obtain detailed biological information. Furthermore, the classification and summary of the modified CLARITY techniques will help researchers to choose a suitable tissue clearing method more quickly and effectively.
In short, passive CLARITY is a right choice for poor laboratory conditions, small samples (tissue thickness < 3 mm), or high requirements for the integrity of tissue structure and molecular information. Of course, passive CLARITY can also be used to clear large samples or even whole mice, but it takes a long time, which puts forward higher requirements for the preservation of tissue and fluorescent staining. The use of active CLARITY can accelerate this process, but the high requirements of laboratory equipment limit its application. In addition, although many improvements have been made to ETC, the tissue damage caused by the current is still the main factor limiting its application. The suitable electric field intensity and electrophoresis time should be strictly selected for different texture tissues.
For the improvement of active CLARITY, the electric field conditions have been optimised in the past, whether active CLARITY can be optimised from its power source. At present, the power that drives lipids to extract from the tissue quickly is electricity, whether it can be replaced with other types of power to avoid tissue damage, such as magnetic energy or hydrodynamic pressure for molecular transport or even centrifugation. Besides, whether active or passive CLARITY, as noted by the researchers who initially developed CLARITY, the subsequent acquisition of high-resolution 3D image and how to analyse these data remains a challenge, which is a common problem in all tissue clearing methods. The improvement of image acquisition and data analysis and exploring how to combine with other technologies will not only make us more skilled in the application of CLARITY but also re-recognise the physiological characteristics of tissue or even a species as a whole for the first time from a 3D perspective.
Acknowledgements
This study was funded by the Science and Technique Innovation Project of Lanzhou University Second Hospital (CY2018-MS14), and the Doctoral Supervisors Research Fund of Lanzhou University Second Hospital (bdkyjj-04).