Advanced CLARITY for rapid and high-resolution imaging of intact tissues

Abstract

CLARITY is a method for chemical transformation of intact biological tissues into a hydrogeltissue hybrid, which becomes amenable to interrogation with light and macromolecular labels while retaining fine structure and native biological molecules. This emerging accessibility of information from large intact samples has created both new opportunities and new challenges. Here we describe next-generation protocols spanning multiple dimensions of the CLARITY workflow, ranging from a novel approach to simple, reliable, and efficient lipid removal without electrophoretic instrumentation (passive CLARITY), to optimized objectives and integration with light-sheet optics (CLARITY-optimized light-sheet microscopy or COLM) for accelerating data collection from clarified samples by several orders of magnitude while maintaining or increasing quality and resolution. These methods may find application in the structural and molecular analysis of large assembled biological systems such as the intact mammalian brain.

Bioimage analysis of Shigella infection reveals targeting of colonic crypts

“…These methods can readily be adapted and applied to other host-pathogen systems, inclusive of the infectious agent, as well as key host factors, including various cell types. Also, the continued development of novel tissue preparation approaches that respect the integrity and three-dimensionality of the tissue, such as optical clearing methods (e.g., CLARITY and CUBIC) (25,26), in combination with continued advancements in imaging techniques, such as light sheet fluorescence microscopy, represent exciting possibilities in the exhaustive extraction of data from pathogeninfected tissues. We hope bioimage analyses will play an increasingly important role in bringing quantitative elements to in vivo host-pathogen studies.…”

Stochastic electrotransport selectively enhances the transport of highly electromobile molecules

“…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).…”

“…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).…”

Novel imaging tools for investigating the role of immune signalling in the brain

“…The physical properties of the lipid bilayer prevents large macromolecules for example, antibodies, from readily diffusing deep into the tissue whilst the molecular heterogeneity of the lipids themselves alter the refractive index of the tissue, further perturbing the excitation and emission of light from fluorescently-labelled probes (Richardson & Lichtman 2015). To overcome these problems, "clearing" methods such as CLARITY, PARS, CUBIC, SeeDB, ScalA2, iDISCO and 3DISCO have been developed (Susaki et al 2015;Bin Yang et al 2014;Renier et al 2014;Tomer et al 2014;Susaki et al 2014;Ke et al 2013;Becker et al 2012;Hama et al 2011). …”

“…However, for optimal staining the incubation time is significantly extended (hours and weeks in tissue slices and whole brains respectively) (Tomer et al 2014) and the working distance of most microscope objectives limits the capture of whole organs. It is worth noting specialized lightsheet microscopes and CLARITY specific objectives can be employed to circumvent this limitation.…”