Three-dimensional imaging of solvent-cleared organs using 3DISCO

Abstract

The examination of tissue histology by light microscopy is a fundamental tool for investigating the structure and function of organs under normal and disease states. Many current techniques for tissue sectioning, imaging and analysis are time-consuming, and they present major limitations for 3D tissue reconstruction. The introduction of methods to achieve the optical clearing and subsequent light-sheet laser scanning of entire transparent organs without sectioning represents a major advance in the field. We recently developed a highly reproducible and versatile clearing procedure called 3D imaging of solvent-cleared organs, or 3DISCO, which is applicable to diverse tissues including brain, spinal cord, immune organs and tumors. Here we describe a detailed protocol for performing 3DISCO and present its application to various microscopy techniques, including example results from various mouse tissues. The tissue clearing takes as little as 3 h, and imaging can be completed in ∼45 min. 3DISCO is a powerful technique that offers 3D histological views of tissues in a fraction of the time and labor required to complete standard histology studies.

Use of labeled tomato lectin for imaging vasculature structures

“…The extensive labeling of vasculature by intravenously applied tomato lectin also opens the possibility of revealing extensive and detailed three-dimensional images of the vasculature of any organ through use of new techniques of tissue clearance (Ertürk et al 2012;Chung et al 2013;Renier et al 2014). …”

Optical clearing of biological tissues: prospects of application in medical diagnostics and phototherapy

“…In comparison with CM or twophoton microscopy, UM can rapidly create optical sectioning of macroscopic samples, since the objectives with small focal power and small numerical aperture can provide a large field of view. However, the use of UM is completely dependent upon the optical transparency of biological objects, so that the improvement of optical clearing efficiency, including that due to the development of new OCAs, remains to be an urgent problem [86,87,[258][259][260][261][262]. In Ref.…”

Stochastic electrotransport selectively enhances the transport of highly electromobile molecules

“…For example, many emerging tissue clearing techniques use surfactant micelles to directly remove lipids from a tissue and thus eliminate light-scattering boundaries to improve optical penetration for holistic visualization (2,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25), but transporting these micelles into the intact tissue via diffusion can take weeks (2,15). Although electrophoresis can speed up this process, as demonstrated in CLARITY, its application has been limited to low electric fields because using high fields can damage tissue structures (2).…”

“…However, with the development of in situ molecular interrogation methods (6,13,14) and tissue clearing techniques (2,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) and an emphasis on studying organ-scale tissue as a whole, a pressing need has arisen for a means of expediting the transportation of various molecules into intact tissues. For example, many emerging tissue clearing techniques use surfactant micelles to directly remove lipids from a tissue and thus eliminate light-scattering boundaries to improve optical penetration for holistic visualization (2,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25), but transporting these micelles into the intact tissue via diffusion can take weeks (2,15).…”

“…However, these techniques have been mostly confined to small samples owing to the difficulty of labeling and examining deep structures in large-scale intact tissues (6,(27)(28)(29)(30)(31). CLARITY and other emerging tissue-clearing techniques (2,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) render intact tissues optically transparent and macromolecule-permeable, allowing examination deep inside Significance Many chemical and biomedical techniques rely on slow diffusive transport because existing pressure-based methods or electrokinetic methods can incidentally damage the sample. This study introduces a novel transport concept termed stochastic electrotransport that can selectively and nondestructively expedite transport of electromobile molecules into a porous sample, such as fixed biological tissues.…”