Light sheet microscopy

Sample clearing – Turning entire samples transparent

Why clear samples?

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Some samples like zebra fish are almost transparent by nature but the majority of samples are opaque counteracting all attempts to explore whole biological systems and processes. Tissue clearing methods render large biological samples transparent while keeping the internal three-dimensional structure. This enables the exploration of whole biological systems and processes using three-dimensional imaging like light sheet microscopy.

Several methods have been published describing different clearing protocols which can be divided into two large groups: organic solvent clearing protocols and water-based clearing protocols.

Organic solvent clearing protocols

The majority of organic solvent clearing procedures has a simple two step mechanism in common: 

  1. Dehydration of the sample by removing water 
  2. Matching refractive indices

The organic solvent clearing leads to very transparent samples and is perfectly suited for dense tissue like tumors, adult tissue, or highly myelinated brain. The majority of immuno-histochemical staining is well conserved. 

Water-based clearing protocols

The most common operating principle of water-based clearing protocols is by depolymerization. By splitting large structures like lipid bilayers into small micelles of different sizes, the opacity is remarkably reduced. Urea can be used as depolimerization agent as with CUBIC clearing. A SDS buffer and an advanced electrophoresis protocol are used for CLARITY clearing. 

The clearing protocols differ in complexity and in the degree of translucency that can be achieved. By depolymerization, the entire structure of a sample can be debilitated while the fluorescence of proteins like GFP is well preserved.

Chung, K. and Deisseroth, K. (2013) CLARITY for mapping the nervous system. Nat. Methods 10(6):508-13

Ertürk, A. et al. (2012) Three-dimensional imaging of solvent-cleared organs using 3DISCO. Nature protocols 7(11):1983-95

Hama, H. et al. (2015) ScaleS: an optical clearing palette for biological imaging, Nat. Neurosci. 18(10):1518-29

Hama, H. et al. (2011) Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat. Neurosci. 14(11):1481-8

Hou, B. et al. (2015) Scalable and DiI-compatible optical clearance of the mammalian brain. Frontiers in neuroanatomy 9:19

Ke, M.T. et al. (2013) SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Nature neuroscience 16(8):1154-61

Ke, M.T. et al. (2016) Super-Resolution Mapping of Neuronal Circuitry With an Index-Optimized Clearing Agent. Cell Rep. 14(11):2718-32

Kim, SY et al. (2015) Stochastic electrotransport selectively enhances the transport of highly electromobile molecules. Proc Natl Acad Sci USA 112(46):E6274-83

Klingberg, A et al. (2016) Fully Automated Evaluation of Total Glomerular Number and Capillary Tuft Size in Nephritic Kidneys Using Lightsheet Microscopy. J Am Soc Nephrol. 28(2):452-459

Kuwajima, T et al. (2013) ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue. Development. 140(6):1364-8

Lee, E et al. (2016) ACT-PRESTO: Rapid and consistent tissue clearing and labeling method for 3-dimensional (3D) imaging. Sci Rep. 6:18631

Murray, E et al. (2015) Simple, scalable proteomic imaging for high-dimensional profiling of intact systems. Cell 163(6):1500-14

Pan, C et al. (2016): Shrinkage-mediated imaging of entire organs and organisms using uDISCO. Nat Methods 13(10):859-67

Renier, N et al. (2014) iDISCO: A Simple, Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging. Cell 159(4):896-910

Schwarz, MK et al. (2015) Fluorescent-Protein Stabilization and High-Resolution Imaging of Cleared, Intact Mouse Brains. PLoS One 10(5):e0124650

Susaki, EA et al. (2015) Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nat Protoc 10(11):1709-27

Susaki, EA et al. (2014) Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell 157(3):726-39

Sylwestrak, EL et al. (2016) Multiplexed Intact-Tissue Transcriptional Analysis at Cellular Resolution. Cell 164(4):792-804

Yang, B et al. (2014) Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell 158(4):945-958

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Insights tissue clearing

Published on Imaging & Microscopy (
Mar. 16, 2017