Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis

Abstract

Systems-level identification and analysis of cellular circuits in the brain will require the development of whole-brain imaging with single-cell resolution. To this end, we performed comprehensive chemical screening to develop a whole-brain clearing and imaging method, termed CUBIC (clear, unobstructed brain imaging cocktails and computational analysis). CUBIC is a simple and efficient method involving the immersion of brain samples in chemical mixtures containing aminoalcohols, which enables rapid whole-brain imaging with single-photon excitation microscopy. CUBIC is applicable to multicolor imaging of fluorescent proteins or immunostained samples in adult brains and is scalable from a primate brain to subcellular structures. We also developed a whole-brain cell-nuclear counterstaining protocol and a computational image analysis pipeline that, together with CUBIC reagents, enable the visualization and quantification of neural activities induced by environmental stimulation. CUBIC enables time-course expression profiling of whole adult brains with single-cell resolution.

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).…”

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). …”