- SeeDB (See Deep Brain) is an optical clearing method that utilizes simple sugar solutions to render biological tissues transparent while maintaining their native structure and fluorescent signals. This technique stands out for its simplicity, rapid clearing time, and minimal tissue deformation, making it particularly valuable for studying delicate biological specimens.
- The fundamental principle of SeeDB involves gradually replacing the water in tissues with fructose solutions of increasing concentration. The final solution matches the refractive index of cellular proteins, reducing light scattering and making the tissue transparent. This approach avoids the use of organic solvents or detergents, helping to preserve the native tissue architecture and molecular content.
- The protocol employs a series of fructose solutions with concentrations ranging from 20% to saturated (approximately 115%). This gradual increase prevents osmotic shock and tissue damage. The final step often includes the addition of α-thioglycerol to prevent browning of the fructose solution, ensuring optimal tissue transparency and long-term stability.
- A key advantage of SeeDB is its exceptional preservation of fluorescent proteins and lipids. Unlike methods that remove lipids or use harsh chemicals, SeeDB maintains membrane structures and protein localization in their original state. This makes it particularly suitable for studying fine cellular structures and protein distributions in intact tissues.
- Sample preparation is straightforward and relatively rapid compared to other clearing methods. Fresh or fixed tissues can be cleared within a few days, depending on size. The process works well with standard fixation protocols and requires no special equipment. This accessibility has made SeeDB popular among researchers studying various tissue types.
- The technique has proven especially valuable in neuroscience research, where it enables detailed examination of neural circuits and synaptic connections. The minimal tissue deformation and excellent preservation of fluorescent proteins make it ideal for studying brain architecture and neuronal relationships at high resolution.
- SeeDB2, an improved version of the original protocol, introduced new clearing agents with higher refractive indices. This modification enables better clearing of myelinated tissues and allows for super-resolution imaging of cleared samples. The enhanced protocol maintains the advantages of the original while expanding its applications.
- One limitation of SeeDB is its effectiveness primarily with smaller tissue samples, as the high viscosity of concentrated fructose solutions can slow diffusion in larger specimens. However, this limitation is often outweighed by the method’s excellent preservation of tissue structure and fluorescent signals.
- Applications of SeeDB extend beyond neuroscience into developmental biology, where it has been used to study embryonic development and organ formation. The method’s ability to maintain tissue integrity while achieving transparency makes it valuable for examining complex developmental processes in three dimensions.
- Integration with modern microscopy techniques, particularly light-sheet microscopy and two-photon imaging, has enhanced the utility of SeeDB-cleared tissues. The optical properties of cleared samples are well-suited for deep tissue imaging, enabling detailed visualization of cellular structures throughout the specimen.
- Data analysis of SeeDB-cleared samples is simplified by the minimal tissue deformation, as spatial relationships remain largely unchanged during the clearing process. This makes it easier to reconstruct three-dimensional structures and analyze cellular relationships without complicated computational corrections.
- Recent developments have focused on optimizing the protocol for specific applications and tissue types. Modified versions have been developed for different imaging modalities and biological questions, while maintaining the core benefits of simplicity and excellent structural preservation.
- The technique has found applications in both research and clinical settings, with protocols adapted for studying human tissue samples. The method’s gentle nature and preservation of tissue architecture make it particularly valuable for analyzing pathological specimens and understanding disease processes.
