ClearT: A detergent- and solvent-free clearing method for neuronal and non-neuronal tissue

Department of Pathology and Cell Biology, Columbia University, College of Physicians and Surgeons, 630 West 168th Street, 14-509 P&S, New York, NY 10032, USA.
Development (Impact Factor: 6.46). 03/2013; 140(6):1364-8. DOI: 10.1242/dev.091844
Source: PubMed


We describe a clearing method for enhanced visualization of cell morphology and connections in neuronal and non-neuronal tissue. Using Clear(T) or Clear(T2), which are composed of formamide or formamide/polyethylene glycol, respectively, embryos, whole mounts and thick brain sections can be rapidly cleared with minimal volume changes. Unlike other available clearing techniques, these methods do not use detergents or solvents, and thus preserve lipophilic dyes, fluorescent tracers and immunohistochemical labeling, as well as fluorescent-protein labeling.

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Available from: Takaaki Kuwajima, Dec 15, 2013
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    • "Recently, tissue optical clearing methods have opened a new avenue to extract cellular resolution information from unsectioned mammalian brains (Dodt et al., 2007; Hama et al., 2011; Becker et al., 2012; Erturk et al., 2012a; Chung et al., 2013; Ke et al., 2013; Kim et al., 2013; Kuwajima et al., 2013; Yushchenko and Schultz, 2013; Renier et al., 2014; Susaki et al., 2014; Tomer et al., 2014; Yang et al., 2014; Zhang et al., 2014). "
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    ABSTRACT: Efficient optical clearance is fundamental for whole brain imaging. In particular, clearance of the brain without membrane damage is required for the imaging of lipophilic tracer-labeled neural tracts. Relying on an ascending gradient of fructose solutions, SeeDB can achieve sufficient transparency of the mouse brain while ensuring that the plasma membrane remains intact. However, it is challenging to extend this method to larger mammalian brains due to the extremely high viscosity of the saturated fructose solution. Here we report a SeeDB-derived optical clearing method, termed FRUIT, which utilizes a cocktail of fructose and urea. As demonstrated in the adult mouse brain, combination of these two highly water-soluble clearing agents exerts a synergistic effect on clearance. More importantly, the final FRUIT solution has low viscosity so as to produce transparency of the whole adult rabbit brain via arterial perfusion, which is impossible to achieve with a saturated fructose solution. In addition to good compatibility with enhanced yellow fluorescent protein, the cocktail also preserves the fluorescence of the lipophilic tracer DiI. This work provides a volume-independent optical clearing method which retains the advantages of SeeDB, particularly compatibility with lipophilic tracers.
    Full-text · Article · Feb 2015 · Frontiers in Neuroanatomy
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    • "Use of this technology allowed for rapid phenotyping and efficient cataloging of defects. Prior to imaging, hearts were fixed in formalin and cleared using the Clear-T protocol, involving incubation of the hearts in a series of increasingly concentrated formamide solutions (Kuwajima et al., 2013). This simple clearing process allowed us to image an entire intact heart at stages after ventricular septation (approximately 2–3 mm thick at stage 35) using OCT. "
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    ABSTRACT: Disturbed cardiac function at an early stage of development has been shown to correlate with cellular/molecular, structural as well as functional cardiac anomalies at later stages culminating in the congenital heart defects (CHDs) that present at birth. While our knowledge of cellular and molecular steps in cardiac development is growing rapidly, our understanding of the role of cardiovascular function in the embryo is still in an early phase. One reason for the scanty information in this area is that the tools to study early cardiac function are limited. Recently developed and adapted biophotonic tools may overcome some of the challenges of studying the tiny fragile beating heart. In this chapter, we describe and discuss our experience in developing and implementing biophotonic tools to study the role of function in heart development with emphasis on optical coherence tomography (OCT). OCT can be used for detailed structural and functional studies of the tubular and looping embryo heart under physiological conditions. The same heart can be rapidly and quantitatively phenotyped at early and again at later stages using OCT. When combined with other tools such as optical mapping (OM) and optical pacing (OP), OCT has the potential to reveal in spatial and temporal detail the biophysical changes that can impact mechanotransduction pathways. This information may provide better explanations for the etiology of the CHDs when interwoven with our understanding of morphogenesis and the molecular pathways that have been described to be involved. Future directions for advances in the creation and use of biophotonic tools are discussed.
    Full-text · Article · Sep 2014 · Frontiers in Physiology
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    • "Cell death during the relatively brief incubation time was very limited. The method of clearing brain tissue with the Clear T2 method (Kuwajima et al., 2013) worked relatively well in auditory brain stem sections, especially in those from younger animals. Fibers in the auditory brain stem myelinate extensively (Ryugo et al., 2006), which greatly impairs imaging quality both for physiological and anatomical studies. "
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    ABSTRACT: Neurons in the medial nucleus of the trapezoid body (MNTB) receive prominent excitatory input through the calyx of Held, a giant synapse that produces large and fast excitatory currents. MNTB neurons also receive inhibitory glycinergic inputs that are also large and fast, and match the calyceal excitation in terms of synaptic strength. GABAergic inputs provide additional inhibition to MNTB neurons. Inhibitory inputs to MNTB modify spiking of MNTB neurons both in-vitro and in-vivo, underscoring their importance. Surprisingly, the origin of the inhibitory inputs to MNTB has not been shown conclusively. We performed retrograde tracing, anterograde tracing, immunohistochemical experiments, and electrophysiological recordings to address this question. The results support the ventral nucleus of the trapezoid body (VNTB) as at least one major source of glycinergic input to MNTB. VNTB fibers enter the ipsilateral MNTB, travel along MNTB principal neurons and produce several bouton-like presynaptic terminals. Further, the contribution of GABA to the total inhibition declines during development, resulting in only a very minor fraction of GABAergic inhibition in adulthood, which is matched in time by a reduction in expression of a GABA synthetic enzyme in VNTB principal neurons.
    Full-text · Article · Jul 2014 · Frontiers in Neural Circuits
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