- CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) is a comprehensive tissue clearing method that uses simple, hydrophilic chemicals to achieve tissue transparency. Developed as an alternative to organic solvent-based and hydrogel-based clearing methods, CUBIC offers a straightforward approach that maintains sample integrity while achieving excellent tissue clarity.
- The CUBIC protocol employs a unique combination of aminoalcohols and detergents to remove lipids and homogenize refractive indices throughout the tissue. The original protocol uses two primary reagents: CUBIC-1 (or Reagent-1), which removes lipids and decolorizes blood by removing heme, and CUBIC-2 (or Reagent-2), which adjusts the refractive index of the tissue to achieve transparency. This two-step approach effectively clears tissues while preserving fluorescent proteins and tissue structure.
- A significant advantage of CUBIC is its compatibility with various fluorescent proteins and immunolabeling techniques. The method preserves most fluorescent proteins exceptionally well, making it particularly valuable for studying transgenic animals expressing fluorescent markers. The cleared tissues remain stable for extended periods, allowing multiple rounds of imaging and analysis.
- CUBIC has proven particularly effective for whole-body clearing of small animals. The technique can render entire mice transparent, enabling researchers to study organ systems and cellular distributions throughout the body. This whole-body approach has provided new insights into system-wide biological processes and disease progression.
- The protocol has evolved with several variations optimized for specific applications. CUBIC-X enables tissue expansion for higher-resolution imaging, while CUBIC-L is optimized for large organ clearing. These modifications have broadened the technique’s utility across different research applications and tissue types.
- Sample preparation is relatively straightforward compared to other clearing methods. Tissues are typically fixed with paraformaldehyde before being immersed in the clearing solutions. The process can be accelerated by gentle shaking and temperature control, though care must be taken to prevent tissue damage. The method works well with both fresh and archived samples.
- One of CUBIC’s strengths is its ability to clear tissues containing high amounts of heme, such as organs with significant blood content. The CUBIC-1 solution effectively removes heme while clearing lipids, making it particularly useful for studying organs like the liver, spleen, and kidneys. This feature has made CUBIC valuable for studying systemic diseases and circulatory system disorders.
- The technique has found broad applications in developmental biology, neuroscience, and cancer research. Researchers have used CUBIC to study embryonic development, map neural circuits, and track cancer cell metastasis. The ability to image entire organs or organisms while maintaining molecular information has provided new perspectives on biological processes.
- Recent developments have focused on optimizing CUBIC for specific tissue types and applications. New reagent formulations have improved clearing speed and tissue preservation while reducing costs. Computational tools have been developed to analyze the large datasets generated from whole-organ imaging, enabling quantitative analysis of cellular distributions and tissue architecture.
- CUBIC has proven particularly valuable in combination with light-sheet microscopy, enabling rapid imaging of large cleared samples. The technique’s compatibility with various microscopy methods and its ability to maintain sample stability during long-term imaging have made it a versatile tool for three-dimensional tissue analysis.
- Applications in human tissue clearing have also emerged, with modified protocols developed for clinical samples. This has enabled new approaches to studying human disease processes and potential therapeutic interventions. The technique’s simplicity and reliability make it particularly suitable for clinical research applications.
- Integration with other techniques has expanded CUBIC’s capabilities. Combinations with single-cell analysis, spatial transcriptomics, and other molecular methods have provided multi-modal insights into tissue organization and function. These integrated approaches continue to reveal new aspects of biological systems and disease processes.
