Extensive mapping of neuronal connections in the central nervous system requires high-throughput µm-scale imaging of large volumes. In recent years, different approaches have been developed to overcome the limitations due to tissue light scattering. These methods are generally developed to improve the performance of a specific imaging modality, thus limiting comprehensive neuroanatomical exploration by multi-modal optical techniques. Here, we introduce a versatile brain clearing agent (2,2′-thiodiethanol; TDE) suitable for various applications and imaging techniques. TDE is cost-efficient, water-soluble and low-viscous and, more importantly, it preserves fluorescence, is compatible with immunostaining and does not cause deformations at sub-cellular level. We demonstrate the effectiveness of this method in different applications: in fixed samples by imaging a whole mouse hippocampus with serial two-photon tomography; in combination with CLARITY by reconstructing an entire mouse brain with light sheet microscopy and in translational research by imaging immunostained human dysplastic brain tissue.

Every optical imaging technique is limited in its penetration depth by scattering occurring in biological tissues. Possible solutions to overcome this problem consist of limiting the detrimental effects of scattering by reducing optical inhomogeneities within the sample. This can be achieved either by using physical methods (such as refractive index matching solutions) or by chemical methods (such as the removal of scatterers), based on tissue transformation protocols. This review provides an overview of the current state-of-the-art methods used for both ex-vivo and in-vivo optical clearing of biological tissues. We start with a brief history of the development of the most widespread clearing methods across the new millennium, then we describe the working principles of both physical and chemical methods. Clearing methods are then reviewed, pointing the attention of the reader on both physical and chemical methods, classified based on the tissue size and type for each specific application. A small section is reserved for methods that have already found in-vivo applications at the research level. Finally, a detailed discussion highlighting both the most relevant results achieved and the new ongoing developments in this field is reported in the last part, together with future perspectives for the clearing methodology.

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Cutting tissue into thin slices has been the standard practice in histology since its early days. Indeed, this approach allows solving (although roughly) two problems at once, i.e., absence of optical sectioning in conventional wide-field microscopes and tissue opacity. However, biological structures are inherently three-dimensional (3-D); for this reason, scientists have always tried to extend tissue imaging to thick specimens. On the imaging side, the invention of confocal microscopy by Minsky in 1955,¹ the development of two-photon microscopy in 1990,² and the recent revival of light-sheet microscopy,³ have provided a wide choice of microscopy methods allowing optical sectioning, i.e., 3-D resolution. On the other hand, the effectiveness and usability of these techniques in real macroscopic specimens are hampered, because biological specimens are naturally opaque.

The distinct organization of the brain’s vascular network ensures that it is adequately supplied with oxygen and nutrients. However, despite this fundamental role, a detailed reconstruction of the brain-wide vasculature at the capillary level remains elusive, due to insufficient image quality using the best available techniques. Here, we demonstrate a novel approach that improves vascular demarcation by combining CLARITY with a vascular staining approach that can fill the entire blood vessel lumen and imaging with light-sheet fluorescence microscopy. This method significantly improves image contrast, particularly in depth, thereby allowing reliable application of automatic segmentation algorithms, which play an increasingly important role in high-throughput imaging of the terabyte-sized datasets now routinely produced. Furthermore, our novel method is compatible with endogenous fluorescence, thus allowing simultaneous investigations of vasculature and genetically targeted neurons. We believe our new method will be valuable for future brain-wide investigations of the capillary network.