This invention can be used for the histological examination of intact tissue in both the research and medical fields, enabling rapid evaluation of healthy and disease states. Specific applications include the identification of neuronal network connections, cell type identities, and long-range projections across organs.
Labeling and imaging of fine structures within complex, intact systems requires sufficient tissue preservation and light penetration. Permeabilizing tissue via lipid removal often results in the dissolving and/or damaging of fine cellular architecture. The use of a fixative prior to controlled electrokinetic delivery of charged macromolecules allows for the detection and binding of intact, fine structures with multi-round capability.
This technology is an electrokinetic device that promotes the rapid flow of charged macromolecules (e.g. detergents, fluorescent probes, antibodies, proteins, nucleic acids) into and out of intact, large-scale biological tissue. A two-step process is used to 1) fix and permeabilize tissue and 2) deliver charged molecules to said tissue. A three-channel design is used whereby the cathode and anode, located in the first and third channels, respectively, lie directly outside two nanoporous membranes that surround the specimen in the second, center channel. These membranes allow for the passage of electric fields and small molecules but restrict macromolecules (e.g. probes of interest) in a size-dependent manner, thus preserving costly reagents and maintaining a steady pH. Multiple probes can be employed at once and separated via pH-based migration. Rotation of the specimen and distribution of electrical force serve to minimize tissue damage. As such, multiple rounds of delivery and extraction are possible in the same tissue sample with minimal damage or protein loss.
- Rapid delivery/extraction of macromolecules: complete lipid removal from intact mouse brain within several hours
Capable of multiple functions
including lipid removal, fluorescent probe/antigen delivery, and nucleic acid
Can perform multiple rounds of
delivery/extraction in an individual tissue sample with minimal damage or
device conserves expensive reagents while controlling pH and temperature