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

By Raju Tomer, Li Ye, Brian Hsueh, Karl Deisseroth. Nature Protocols, 2014

810 citations recorded by the source corpus.

CLARITY is a method for chemical transformation of intact biological tissues into a hydrogel-tissue 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 protocols spanning multiple dimensions of the CLARITY workflow, ranging from 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 (COLM)) for accelerating data collection from clarified samples by several orders of magnitude while maintaining or increasing quality and resolution. The entire protocol takes from 7–28 d to complete for an adult mouse brain, including hydrogel embedding, full lipid removal, whole-brain antibody staining (which, if needed, accounts for 7–10 of the days), and whole-brain high-resolution imaging; timing within this window depends on the choice of lipid removal options, on the size of the tissue, and on the number and type of immunostaining rounds performed. This protocol has been successfully applied to the study of adult mouse, adult zebrafish and adult human brains, and it may find many other applications in the structural and molecular analysis of large assembled biological systems.

Subfields: Biophysics, Structural Biology

View Paper: https://www.semanticscholar.org/paper/aec60672e5ee9edd8606a5d2949e17dfbd01c863

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Citations in the corpus

Simple, scalable proteomic imaging for high-dimensional profiling of intact systems (2015, positive): “We imaged this larger volume using a custom-built, highspeed light-sheet microscope (Tomer et al., 2012; Tomer et al., 2014) within 2 hours and observed uniform labeling of all myelinated fibers across the entire tissue (Figure 5J; Movie S4).”
Closed-loop and activity-guided optogenetic control. (2015, positive): “…Smith, 2007) and wiring information (see circuit-targeting strategies reviewed below and Bock et al., 2011) could be further obtained (for example, leveraging recent advances in multiround molecular and anatomical analysis in large, intact cleared brains; Chung et al., 2013; Tomer et al., 2014).”
Intact-Brain Analyses Reveal Distinct Information Carried by SNc Dopamine Subcircuits (2015, methodology): “…CLARITYmonomer solution containing 1% acrylamide, 0.0125% bis-acrylamide, and 4% PFA and then polymerized at 37 C for 6–7 hr. Brains were passively cleared in SDS Borate Buffer (pH 8.5) at 37 C for 4–5 weeks, equilibrated in Focusclear for imaging and imaged using COLM methods (Tomer et al., 2014).”
iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. (2014, neutral): “The advantages of light-sheet microscopy over point-scanning microscopy are 908 Cell 159, 896–910, November 6, 2014 ª2014 Elsevier Inc. reduced sample bleaching, high acquisition speed, large fields of view, and high dynamic range (Tomer et al., 2014).”
Wiring and Molecular Features of Prefrontal Ensembles Representing Distinct Experiences (2016, neutral): “The COLM system (described earlier in Tomer et al., 2014; Lerner et al., 2015) is optimized for high-resolution imaging and is most useful for studies requiring sub-cellular information, while the approach outlined here is optimized for speed and practicality in cellular resolution work, most…”
Clarifying Tissue Clearing (2015, positive): “Lightsheet microscopes utilizing these designs are optimal for cleared tissue imaging when the highest optical resolution is not essential (Dodt et al., 2007; Susaki et al., 2014; Tomer et al., 2014; Verveer et al., 2007).”
Single-Cell Phenotyping within Transparent Intact Tissue Through Whole-Body Clearing (2014, positive): “RESULTS Optimized Method for Passive Clearing and Immunostaining of Whole Organs in the Rodent Similar to the CLARITY method (Chung et al., 2013; Tomer et al., 2014), we render thick tissue optically transparent for imaging in three main steps.”
NeuroStorm: Accelerating Brain Science Discovery in the Cloud (2018, methodology): “One application demonstrating the registration of a CLARITY [24] image to the Allen Reference Atlas [25] and parcellation at 50um resolution is shown in Fig.”