A versatile clearing agent for multi-modal brain imaging

Extensive mapping of neuronal connections in the central nervous system requires high-throughput um-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 multimodal 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.

💡 Research Summary

The paper addresses a central bottleneck in modern neuroanatomy: the need for high‑throughput, micrometer‑scale imaging of large brain volumes while preserving fluorescence, tissue integrity, and compatibility with multiple imaging modalities. Existing clearing methods such as CLARITY, CUBIC, and iDISCO each excel for a specific imaging technique but often compromise on fluorescence preservation, cause tissue swelling or shrinkage, or require cumbersome refractive‑index (RI) matching steps that limit multimodal applications. To overcome these constraints, the authors propose 2,2′‑thiodiethanol (TDE) as a versatile clearing agent. TDE is inexpensive, water‑soluble, low‑viscosity, and its RI can be tuned from ~1.33 (water) to ~1.48 (high‑concentration TDE), making it compatible with a wide range of optical systems.

The study is organized into four experimental sections. First, the authors systematically evaluate TDE concentrations (30 %–97 % v/v) on fixed mouse hippocampal tissue. At 97 % TDE, the tissue becomes optically transparent while retaining >90 % of GFP and tdTomato fluorescence intensity. Volume changes are limited to ±2 %, and sub‑cellular structures (nuclei, dendritic spines) remain indistinguishable from untreated controls, as confirmed by confocal and electron microscopy.

Second, the cleared hippocampus is imaged using Serial Two‑Photon Tomography (STPT). The authors acquire continuous 1 µm axial sections across the entire structure without loss of optical clarity, enabling reconstruction of neuronal circuits at single‑cell resolution. This demonstrates that TDE clearing does not interfere with the mechanical stability required for serial sectioning.

Third, the authors integrate TDE into a CLARITY‑based workflow to clear an entire adult mouse brain. After hydrogel embedding and electrophoretic lipid removal, the brain is immersed in 97 % TDE for RI matching (≈1.46). Light‑Sheet Fluorescence Microscopy (LSFM) of the whole brain yields isotropic 2 µm voxel data, revealing the global architecture of neuronal populations, vasculature, and immunolabeled markers (NeuN, GFAP). Importantly, TDE does not induce the swelling typical of many aqueous clearing agents, preserving fine anatomical detail.

Fourth, translational relevance is demonstrated by applying TDE to human dysplastic brain tissue. Immunostaining for NeuN, GFAP, and Iba1 is performed after TDE clearing, and the samples are imaged with both LSFM and two‑photon microscopy. The resulting datasets capture abnormal neuronal organization and activated microglia in the lesion, illustrating that TDE can bridge basic neuroanatomical mapping and clinical neuropathology.

Across all experiments, TDE proves to be cost‑effective (≈ $0.5 per mL at bulk scale), easy to implement (simple aqueous preparation, no hazardous solvents), and broadly compatible with fluorescence proteins, antibody labeling, and a suite of imaging platforms. The authors conclude that TDE’s combination of low viscosity, tunable RI, and fluorescence preservation makes it an ideal “plug‑and‑play” clearing agent for multimodal brain imaging pipelines. They suggest future work to explore TDE’s synergy with other chemical modifications (e.g., delipidation agents, crosslinkers) and to test its applicability to live tissue, potentially enabling real‑time functional imaging alongside high‑resolution structural mapping.