Abstract
The article reflects on the development of tissue clearing methods in the last decade. This technological innovation has been achieved in the neuroscience community thanks to the combined efforts of molecular biologists, brain anatomists and light microscopists. Although this technology is becoming increasingly diversified, it mostly aims at the large-scale, three-dimensional reconstruction of brain structures.
With the advent of two-photon excitation fluorescence microscopy (TPEFM) and genetic methods for fluorescent labeling, life scientists await a new optical technique that could provide both the fine and broad perspectives of labeled structures within a large biological specimen. In the neuroscience community, it is very difficult to produce high-resolution reconstructions of entire neuronal networks in fixed brain samples where specific cell types are labeled with fluorescent proteins. Because of this, there is a high demand for new techniques that seek to address this issue, such as the development of Brainbow mice [1]. Such tools are critical for comprehensive ‘connectomic analyses’ [2,3].
The three-dimensional (3D) imaging of large biological specimens requires sectioning in order to improve axial resolution. It is also necessary to achieve subcellular resolution for the 3D reconstruction of fluorescently labeled structures within such large tissue samples as whole mouse brains. For example, the cerebral cortex of the adult mouse brain is ∼1 mm thick. Accordingly, we need to examine tissue specimens of millimeter-order thickness in order to comprehensively survey fluorescently labeled neuronal circuits. On the one hand, mechanical sectioning methods allow for the efficient observation of genetically or immunohistochemically labeled structures with subcellular resolution, but are extremely laborious for large-scale 3D reconstruction in the absence of such well-designed automation procedures as array tomography [4]. 3D reconstruction using electron microscopy (EM) ensures nano-scale resolution, but EM is generally applicable to very small specimens. Although recently developed techniques have improved the quality of volume EM data and automated the data acquisition process [5,6], it remains to be seen whether the techniques can efficiently process large tissue volumes. On the other hand, optical sectioning methods are highly promising, but optical imaging deep into tissue is prevented mostly by light scattering. Generally, a biological sample can only be viewed to a depth of ∼150 μm from the surface by laser scanning confocal microscopy. To overcome the problem stemming from light scattering, tissue clearing technology aims to increase tissue transparency in order to achieve refractive uniformity throughout a fixed sample. This technique basically involves the incubation of fixed brain samples in a clearing reagent for some time.
Two types of tissue clearing reagents are available. First, organic chemical based solutions are highly capable of optically clearing fixed samples. A research group dehydrated fixed samples with ethanol and hexane, and then incubated the samples in a clearing solution of one part benzyl alcohol and two parts benzyl benzoate (abbreviated as BABB) [7,8]. It was pointed out, however, that the chemical clearing procedure substantially quenched fluorescent proteins inside the samples. To solve the problem, the group replaced alcohol with tetrahydrofuran (THF) in combination with BABB. THF- and BABB-based clearing was also effective for clearing myelinated tissue, such as the spinal cord [9]. Furthermore, BABB was replaced with dibenzyl ether (DBE) to develop a THF- and DBE-based solution that was applicable to the adult brain. These techniques are called 3DISCO (3D imaging of solvent-cleared organs) [10]. In 2014, another research group simplified the 3DISCO procedure and established a simple, rapid and inexpensive method, iDISCO, which permits whole-mount immunolabeling with volume imaging of large cleared samples [11]. Its compatibility with 28 antibodies to both endogenous antigens and transgenic reporters, such as green fluorescent protein, was demonstrated.
Second, quite a few aqueous solutions have been developed as well. FocusClear has been successfully used to clear whole brains of insects, including cockroach and fruit fly. Unfortunately, this commercial reagent is prohibitively expensive [12–14]. Furthermore, because its contents are undisclosed, the properties of FocusClear are unknown, which makes it difficult to optimize clearing procedures for different biological samples. Although the use of FocusClear was limited mostly to entomological studies, recent studies with CLARITY (below) showed that the reagent sufficiently cleared the brain tissues of mammals. In 2009, combined techniques were reported for labeling and clearing thick slabs of mouse cortex [15]. The slabs were cleared by immersion in an index matching solution of sucrose (60% w/v), and vasculature and nuclei were imaged at depths of ∼1.2 mm with TPEFM. However, the sucrose clearing method provides only modest transparency to the tissue.
In my group, the discovery of the Scale reagent started with the serendipitous observation that polyvinylidene fluoride membranes became transparent when soaked in 4 M urea, which promotes the hydration of biological samples. This result inspired us to search for an optimal aqueous reagent to clear fixed biological samples for light microscopy. We experimented with various concentrations of urea and other ingredients and found that the most effective solution (ScaleA2) was composed of 4 M urea, 10% glycol and 0.1% Triton X-100. With the Scale system, in 2011, we presented a simple but powerful technique for clearing mammalian brain tissue, which has the potential to address questions in brain structure and function at an unprecedented spatial scale and detail (Fig. 1) [16].
Three-dimensional reconstruction of yellow fluorescent protein-expressing neurons in a quadratic prism of the mouse hippocampus cleared using ScaleA2. Distribution of neurons and projections of their neurites are shown in a large brain volume at a high spatial resolution. DG, dentate gyrus.
Since 2012, there has been a revival of interest in optimizing and improving techniques for large-scale 3D imaging of molecularly labeled structures in cleared tissue samples. That has led to the development of several important new clearing methods with aqueous solutions. The CLARITY method involves fixation as well as clearing of tissue [17]. First, tissue is cross-linked and hybridized to hydrogel monomers for protein stabilization. Then, tissue lipids are extracted from the tissue–hydrogel matrix with detergents. This lipid extraction is performed electrophoretically in the original form of CLARITY. However, in recent modified versions of CLARITY, such as advanced CLARITY [18] and passive clarity technique (PACT) [19], this active procedure (electrophoretic tissue clearing, ETC) has been replaced with a passive one. The PACT method includes a refractive index matching solution (RIMS), which is the alternative to FocusClear. In addition, PACT reagents are delivered either intracranially or via the vasculature; this procedure is termed PARS (perfusion-assisted agent release in situ). ClearT [20] and SeeDB [21] use formamide and fructose, respectively, and both show very good preservation of fluorescent signals despite their moderate clearing capability. Although clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) uses both urea and Triton X-100 and is similar to ScaleA2, it employs amino alcohol as a very critical component instead of glycerol [22]. CUBIC shows very powerful tissue clearing capability and is suitable for whole-brain and whole-body clearing [23].
An increasing number of studies are expected to be conducted to assess the strength and limitations of those methods, compare their performances, provide solutions to extend their range of utility and discuss their potential roles in emerging connectomics efforts to map the brain and its constituent structures. Given their simplicity and/or stability, I believe those novel methods will popularize high-resolution 3D reconstructions within mammalian brain and other tissues, organs and animals.
Optical clearing methods have co-evolved with the advances in hardware and software. For example, whole-brain or whole-body imaging has made full use of light-sheet microscopy (ultramicroscopy). The emergence of new tissue clearing methods will no doubt stimulate the imagination of many neuroscientists. The demand for such methods and the aspirations of researchers who use them are sure to soar. As a result, fluorescence microscopes will inevitably have to be equipped with special hardware and software to make the best use of them. In this regard, a significant evolution of light microscopes will be necessary if large-scale 3D reconstructions are to enjoy widespread use. Commercial light microscopy systems should evolve into ones that are amenable to the addition of new functions.
Acknowledgements
This work was partly supported by the Brain/MINDS (Mapping by Integrated Neurotechnologies for Disease Studies).
