- DISCO (3DISCO, iDISCO, uDISCO) represents a family of organic solvent-based tissue clearing methods that have revolutionized the way researchers visualize large biological specimens. These techniques achieve tissue transparency through dehydration and refractive index matching using organic solvents, enabling deep tissue imaging while preserving cellular and molecular information.
- The original 3DISCO method introduced a three-dimensional imaging of solvent-cleared organs approach, using a combination of tetrahydrofuran (THF) for dehydration and dibenzyl ether (DBE) for refractive index matching. This protocol effectively clears tissues but had limitations regarding fluorescence preservation and long-term sample storage. These challenges led to the development of improved variants.
- iDISCO (immunolabeling-enabled DISCO) enhanced the original protocol by optimizing it for immunolabeling applications. This variant introduced methanol-based tissue pretreatment steps that improve antibody penetration and reduce autofluorescence. iDISCO enables whole-mount immunolabeling of large tissue samples, making it particularly valuable for studying protein distribution in intact organs.
- uDISCO (ultimate DISCO) addressed the fluorescence preservation issues of earlier methods by introducing new chemical combinations that better preserve fluorescent proteins and reduce tissue shrinkage. This variant uses tert-butanol for dehydration and BABB-D (a mixture of benzyl alcohol, benzyl benzoate, and diphenyl ether) for refractive index matching, resulting in improved sample stability and fluorescence retention.
- A key advantage of DISCO methods is their rapid clearing speed compared to aqueous-based techniques. Large samples can be cleared within days rather than weeks or months. The significant tissue shrinkage that occurs during clearing, while sometimes considered a limitation, can actually be advantageous for imaging very large specimens, as it brings more tissue within the imaging volume of standard microscopes.
- Sample preparation for DISCO methods requires careful attention to proper tissue fixation and handling of organic solvents. The protocols typically begin with paraformaldehyde fixation, followed by careful dehydration steps to avoid tissue damage. Proper ventilation and safety measures are essential due to the use of organic solvents.
- These methods have found particular application in neuroscience research, enabling the study of neural circuits, cellular distributions, and protein expression patterns throughout entire brains. The ability to image large tissue volumes while maintaining cellular resolution has provided new insights into brain organization and function.
- DISCO techniques have also proven valuable in developmental biology and cancer research. They enable visualization of embryonic development, organ formation, and tumor metastasis in intact specimens. The methods’ compatibility with various labeling techniques allows researchers to track specific cell populations and molecular markers throughout entire organisms.
- Recent developments have focused on improving tissue preservation, reducing clearing time, and enhancing compatibility with various labeling methods. New solvent combinations and protocol modifications continue to emerge, each optimized for specific applications or tissue types. These improvements have made DISCO methods more versatile and reliable.
- The integration of DISCO clearing with advanced imaging technologies, particularly light-sheet microscopy, has enabled rapid acquisition of high-resolution data from large cleared samples. Specialized imaging chambers and objectives have been developed to accommodate samples cleared with organic solvents, optimizing image quality and data collection.
- Data analysis presents significant challenges due to the large datasets generated from whole-organ imaging. Specialized software tools have been developed to handle these datasets, enabling three-dimensional reconstruction, automated feature detection, and quantitative analysis. The tissue shrinkage characteristic of DISCO methods must be accounted for in these analyses.
- Applications continue to expand beyond traditional research settings into clinical applications. Modified protocols have been developed for human tissue samples, enabling new approaches to studying disease processes and potential treatments. The rapid clearing speed of DISCO methods makes them particularly attractive for clinical research applications where time is often critical.
