2.1.

Organic Solvents

The first clearing method for fixed tissue, reported by Spalteholz a hundred years ago,²⁰ was based on the substitution of water with a mixture of benzyl benzoate and methyl salicylate, showing a refractive index of about 1.55. Since benzyl benzoate is insoluble in water, an intermediate dehydration step based on ethanol was introduced. This general scheme (dehydration followed by incubation in high-refractive index solvents) has been reintroduced by Dodt et al.⁸ who exploited as index-matching solution a mixture of benzyl alcohol and benzyl benzoate (BABB, $n=1.55$). Since the original BABB protocol quenches the emission of fluorescent proteins after a short time period, the same group investigated alternative dehydration and clearing agents, finding that dehydration with tetrahydrofuran (THF) and clearing in dibenzyl ether (DBE, $n=1.56$) achieved the best results in fluorescence preservation and sample transparency (3DISCO).^6,9 Notably, the authors pointed out that both THF and DBE tend to develop fluorescence-quenching peroxides, and for this reason all solutions should be previously filtered in alumina.⁶

Other improvements on BABB clearing, based on pH control and different dehydration agents, have been recently reported by Schwarz et al. (FluoClearBABB).²⁶ A further extension of clearing based on organic solvents is represented by the embedding of samples in clear, organic-based, solid resins: an intriguing feature of this method is the large reduction of photobleaching.¹¹

2.2.

High-Refractive Index Aqueous Solutions

Fluorescence can be more easily preserved if the sample is maintained in an aqueous, protein-friendly environment. Indeed, fluorescence quenching observed in organic solvents (at least in some protocols) stimulated the quest for water-based solutions with high refractive index. Some methods exploit high-concentration solutions of sugars, such as sucrose ($n=1.44$)²⁵ or fructose (SeeDB and FRUIT, $n=1.48$).^12,13 A drawback of sugar solutions is their high viscosity; to solve this problem, alternative approaches based on organic compounds like 2,2′-thiodiethanol (TDE, $n=1.42$)^19,23,28 have been proposed. The family of water-based clearing includes also commercial solutions whose detailed composition is not known (FocusClear, $n=1.45$).

2.3.

Hyperhydrating Solutions

In general, the index of refraction of aqueous solutions cannot be increased above 1.48. However, this is usually not enough to correctly match the average refractive index of the dry component of the tissue.⁴ A workaround to this problem is to simultaneously act on tissue components (such as proteins) to lower their effective refractive index, and/or to remove other components such as lipids. Indeed, lipid removal exploiting a polyalcohol (glycerol) and a detergent (Triton X-100), and protein hyperhydration with urea are the main building blocks of the Scale method ($n=1.38$).⁵ The results obtained with Scale triggered a more comprehensive screening of chemical agents (polyalcohols, detergents, and hydrophilic compounds), which resulted in a protocol called CUBIC ($n=1.48$).²² CUBIC has been recently further optimized to afford clearing of the whole mouse body, guaranteeing also discoloration of hemoglobin.²⁴ A further development of Scale is ScaleS, a method where glycerol is replaced by sorbitol.²⁷

Another protocol exploiting hyperhydration (without lipid removal) is based on formamide/water mixtures (${\text{Clear}}^{\mathrm{T}}$, $n=1.44$).²¹

2.4.

Tissue Transformation

Although removal of lipids from the tissue does provide increased transparency at moderate refractive indexes ($<1.48$, affordable by aqueous solutions), the use of strong detergents results also in a loss of protein content from the tissue. Furthermore, strongly hydrophilic molecules like urea can lead to protein denaturation, hindering epitopes potentially useful for IHC. Tissue transformation techniques try to stabilize the protein content of the sample by cross-linking proteins to a gel mesh. The pioneer methodology in the field has been CLARITY, where proteins and nucleic acids are cross-linked to a polyacrylamide gel by paraformaldehyde.¹⁴ The tissue–gel hybrid is then treated with a strong detergent [sodium dodecyl sulfate (SDS)] for lipid removal. Finally, the sample is immersed in an index-matching solution (originally FocusClear) to render it transparent.

Lipid removal by SDS can be sped up by electrophoresis¹⁴ or by raising incubation temperature.¹⁵ However, strong electric field or high temperatures can damage tissue–gel hybrids on both a structural and molecular level. Stochastic implementations of electrophoresis¹⁶ and novel tissue-hybrid recipes¹⁷ improve sample stability while keeping lipid removal time quite short (few days). Continuous perfusion of the sample with SDS is another approach to improve clearing speed.¹⁸ Since the same perfusion system can be used also to deliver hydrogel monomers as well as immunohistochemical solutions (like antibodies and blocking buffers), this approach has been termed perfusion-assisted agent release in situ (PARS).¹⁸ Other improvements to the original CLARITY approach include the use of cheaper index matching solutions, such as TDE,¹⁹ histodenz,¹⁸ or diatrizoic acid.^16,17

Tissue-transforming methods are usually quite complex and expensive; nonetheless, they are attractive because of the high degree of transparency and of the increased sample porosity, which helps whole-mount IHC on large portions of tissue.^14,19 Diffusion of antibody inside the sample can be further facilitated by the application of stochastic electric fields.¹⁶

3.1.

Whole Organ Imaging with Light-Sheet Microscopy

Providing optical sectioning in a wide-field detection scheme, light-sheet microscopy (LSM) is one of the fastest methods for volumetric imaging.³ Planar illumination can be provided either by cylindrical optics²⁹ or by one-dimensional scanning of a line.³⁰ The excitation beam is poorly focused, guaranteeing that the thickness of the light sheet is almost constant within the field of view of the camera. Visible light is generally used, although two-photon LSM has been demonstrated for small samples.³¹ The linear excitation mechanism makes this technique quite sensitive to light scattering inside the sample, which results in unwanted blur and eventually in a completely misty image. Therefore, although some optical methods to reduce scattering effects have been devised in the last years (like confocal detection^32,33 and Bessel beam illumination³⁴), LSM requires the sample to be as transparent as possible for proper imaging. Indeed, LSM of macroscopic biological specimens was coupled to tissue clearing since its first reported application in 1993.³⁵

When choosing the appropriate clearing method for LSM, the most important feature to be taken into account is a complete, crystalline, clearing. “Average” transparency usually does not work. Here below, we list published clearing protocols suitable for LSM, according to their compatibility with endogenous fluorescence (e.g., GFP) and/or ex vivo tissue staining. A visual summary of the clearing and imaging capabilities of the various methods is reported in Fig. 3.

Fig. 3

Whole-brain transparencies and representative fluorescence images obtained with different methods. Images adapted with permission from Refs. 14, 1617.18.–19, 22, and 36.

3.2.

Large Area Imaging with Two-Photon or Confocal Microscopy

Confocal and two-photon fluorescence microscopy (TPFM) are probably the most common imaging methods capable of optical sectioning (i.e., volumetric imaging).^40–42 Typical setups are based on epi-fluorescence detection and a scanning system (usually a pair of galvo mirrors) to reconstruct the image of the sample point by point. Optical sectioning capabilities of both methods are proportional to the NA of the objective used, and in practice moderate to large numerical apertures ($\mathrm{NA}>0.5$) are used. This fact contributes to render confocal and two-photon microscopy sensible to optical aberrations.⁴³ Thus, in general, these techniques require objective lenses corrected for the refractive index of the clearing solution employed.

The sequential acquisition scheme usually employed in confocal and two-photon microscopy limits imaging speed,³⁶ restricting the common use of these methods only portions of entire organs.¹⁹ To afford full reconstruction of whole mouse brains, significant engineering efforts are required to keep system stability over long (several weeks) imaging times.^44,45 These stability requirements also apply to the sample itself; if cm-sized samples have to be imaged with point-scanning methods, the clearing method used must provide long-term sample stability and fluorescence preservation.

TPFM is known to perform well deep inside scattering tissue.⁴² Indeed, due to the nonlinearity of excitation, diffused light does not contribute to background signal since it cannot reach enough intensity to excite fluorescence; in addition, the near-infrared wavelengths used for excitation are scattered less by biological specimens. TPFM is nowadays the standard method for in vivo imaging deep (up to 700 to $800\mu \mathrm{m}$) in mouse brain. However, the penetration depth of this method in aldehyde-fixed tissue is reduced to about $300\mu \mathrm{m}$ because of increased optical scattering.¹² Nonetheless, the robustness to scattering intrinsic to TPFM allows its use for volumetric imaging also in poorly cleared specimens.

To summarize, when choosing the appropriate clearing protocol for confocal or two-photon microscopy, the main requirement concerns the availability of suitable objective lenses, while for TPFM high transparency is not as crucial as for LSM. From these considerations, we can conclude that high-refractive-index organic solvents are not the best choice in this case, while direct immersion in aqueous solution can be a valid approach, although it provides only moderate transparency. Finally, if one wants to exploit serial tissue sectioning to perform reconstructions on larger areas, also tissue stability and cutting capability become important and should be taken into account.

Here below, we list published clearing protocols suitable for confocal and two-photon microscopy, according to their compatibility with endogenous fluorescence (e.g., GFP) and/or ex vivo tissue staining. A visual summary of the clearing and imaging capabilities of the various methods is reported in Fig. 4.

Fig. 4

Brain slice transparencies and representative fluorescence images obtained with different methods. Images adapted with permission from Refs. 5, 12, 19, 21, and 27.