Biology:CUBIC

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CUBIC (abbreviation for “clear, unobstructed brain/body imaging cocktails and computational analysis)[1] is a histology method that allows tissues to be transparent (process called “tissue clearing”). As a result, it makes investigation of large biological samples with microscopy easier and faster. The method was published in 2014 by Etsuo A. Susaki and Hiroki R. Ueda, primarily for use in neurobiology research of brains from model organisms like rodents or small primates.[1] But in upcoming years there were other works published, using CUBIC method on other tissues like lymph nodes[2] or mammary glands.[3] CUBIC can be also combined with CLARITY-based tissue clearing methods.[1][4]

Used chemicals and performing of method

The opacity of brain tissue is given mainly by light scattering on interfaces between environments with various refractive indexes, mainly between lipids and other tissue compounds.[5] Therefore, partial delipidating and refractive index matching of tissue and surrounding medium is straightforward way to make tissue less opaque and therefore transparent – cleared.[1]

Development of CUBIC pipeline was inspired by previously published clearing protocol named Scale (mixture of glycerol, urea and detergent), because of its simplicity and optimal compatibility with fluorescent proteins.[6] Authors of CUBIC screened 40 chemicals corresponding to those used in Scale with aim to conserve compatibility with fluorescent reporters but achieve better and faster clearing of the tissue. They found that basic amino alcohols are ideally suited for this purpose, probably because amino groups effectively solvate phospholipids and basicity helps to preserve fluorescence signal.[1] Amino alcohols have also beneficial effect when used for clearing of other tissues, which are mostly highly vascularized, and their opacity is given by absorption of light by hemoglobin on top of light scattering. Amino alcohols reduce pigmentation of those tissues very effectively by eluting the hem from hemoglobin.[7]

The original protocol[7] is two-step incubation of fixed tissue in two different aqueous based clearing solutions, altogether taking one to two weeks. First solution, referred as ScaleCUBIC-1, CUBIC-1 or just reagent-1, is composed of N,N,N’,N’-tetrakis(2-hydroxypropyl)ethylenediamine (commercially under name Quadrol), urea and Triton X-100 in water. Second solution, referred as ScaleCUBIC-2, CUBIC-2 or just reagent-2, is composed of urea and sucrose in water.[8] This original protocol is slightly modified in different applications, namely in concentrations, incubation times or some components of solutions.[7][9][10] The CUBIC protocol can be also combined with perfusion and provide whole organ and whole body clearing of rodents.[7] Besides its use as standalone protocol for clearing, CUBIC-based composition of reagents can be used as refractive index matching solution for CLARITY.[4] This than improve clearing abilities of CLARITY on tissues which stay opaque because of their pigmentation by hemoglobin.

The improvement of CUBIC reagents has progressed and been reported.[11]

Applications of method

The CUBIC method is very powerful, due to amino alcohols and consequent ability to clear almost any organ or even whole body of mice.[7] One disadvantage is incompatibility with lipid dyes, due to high concretion of detergent used during clearing.[12] Despite organic solvents, reagents used in CUBIC are not toxic or aggressive to optics of microscopes,[5] on the other hand the handling with them can be also tricky due to their high viscosity. Also, in comparison with solvent based methods like 3DISCO the CUBIC as method based on simple diffusion is still take slightly longer times for clearing same tissue.[13][5]  Because of high concentration of detergent, the CUBIC ca also partially diminishes the fluorescence or disturb ultrastructure of the tissue.[14]

CUBIC was optimized and used for wide spread of applications and tissues. In the mouse the method was used for mapping the brain activity,[14] analysis of interactions between immune cells in lymph node,[2] description of mammary stem cells behaving[3] or for capturing 3D anatomy of liver, kidneys, lungs and heart.[7] CUBIC was also modified and used for clearing of chicken embryos[15] or marmoset brains.[1] The method was also optimized for clearing and examination of tumors and development of metastases from whole body perspective to single-cell resolution in mouse model.[16] In recent study was also shown few variants of basic CUBIC pipeline, in this case used for diagnostic of human pathologies both on native and formalin fixed paraffin embedded tissues.[10]

See also

References

  1. ↑ 1.0 1.1 1.2 1.3 1.4 1.5 Susaki, Etsuo A.; Tainaka, Kazuki; Perrin, Dimitri; Kishino, Fumiaki; Tawara, Takehiro; Watanabe, Tomonobu M.; Yokoyama, Chihiro; Onoe, Hirotaka et al. (2014). "Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis". Cell 157 (3): 726–739. doi:10.1016/j.cell.2014.03.042. PMID 24746791. 
  2. ↑ 2.0 2.1 Abe, Jun; Ozga, Aleksandra J.; Swoger, Jim; Sharpe, James; Ripoll, Jorge; Stein, Jens V. (2016). "Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodes". Journal of Immunological Methods 431: 1–10. doi:10.1016/j.jim.2016.01.015. PMID 26844990. 
  3. ↑ 3.0 3.1 Davis, Felicity M.; Lloyd-Lewis, Bethan; Harris, Olivia B.; Kozar, Sarah; Winton, Douglas J.; Muresan, Leila; Watson, Christine J. (2016-10-25). "Single-cell lineage tracing in the mammary gland reveals stochastic clonal dispersion of stem/progenitor cell progeny". Nature Communications 7: 13053. doi:10.1038/ncomms13053. PMID 27779190. Bibcode: 2016NatCo...713053D. 
  4. ↑ 4.0 4.1 Lee, Eunsoo; Choi, Jungyoon; Jo, Youhwa; Kim, Joo Yeon; Jang, Yu Jin; Lee, Hye Myeong; Kim, So Yeun; Lee, Ho-Jae et al. (2016-01-11). "ACT-PRESTO: Rapid and consistent tissue clearing and labeling method for 3-dimensional (3D) imaging". Scientific Reports 6 (1): 18631. doi:10.1038/srep18631. ISSN 2045-2322. PMID 26750588. Bibcode: 2016NatSR...618631L. 
  5. ↑ 5.0 5.1 5.2 Richardson, Douglas S.; Lichtman, Jeff W. (2015). "Clarifying Tissue Clearing". Cell 162 (2): 246–257. doi:10.1016/j.cell.2015.06.067. PMID 26186186. 
  6. ↑ Hama, Hiroshi; Kurokawa, Hiroshi; Kawano, Hiroyuki; Ando, Ryoko; Shimogori, Tomomi; Noda, Hisayori; Fukami, Kiyoko; Sakaue-Sawano, Asako et al. (November 2011). "Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain". Nature Neuroscience 14 (11): 1481–1488. doi:10.1038/nn.2928. ISSN 1546-1726. PMID 21878933. 
  7. ↑ 7.0 7.1 7.2 7.3 7.4 7.5 Tainaka, Kazuki; Kubota, Shimpei I.; Suyama, Takeru Q.; Susaki, Etsuo A.; Perrin, Dimitri; Ukai-Tadenuma, Maki; Ukai, Hideki; Ueda, Hiroki R. (2014). "Whole-Body Imaging with Single-Cell Resolution by Tissue Decolorization". Cell 159 (4): 911–924. doi:10.1016/j.cell.2014.10.034. PMID 25417165. 
  8. ↑ Susaki, Etsuo A; Tainaka, Kazuki; Perrin, Dimitri; Yukinaga, Hiroko; Kuno, Akihiro; Ueda, Hiroki R (November 2015). "Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging". Nature Protocols 10 (11): 1709–1727. doi:10.1038/nprot.2015.085. ISSN 1750-2799. PMID 26448360. https://eprints.qut.edu.au/88909/3/88909.pdf. 
  9. ↑ Nehrhoff, Imke; Bocancea, Diana; Vaquero, Javier; Vaquero, Juan José; Ripoll, Jorge; Desco, Manuel; Gómez-Gaviro, María Victoria (2016-09-01). "3D imaging in CUBIC-cleared mouse heart tissue: going deeper". Biomedical Optics Express 7 (9): 3716–3720. doi:10.1364/boe.7.003716. ISSN 2156-7085. PMID 27699132. 
  10. ↑ 10.0 10.1 Nojima, Satoshi; Susaki, Etsuo A.; Yoshida, Kyotaro; Takemoto, Hiroyoshi; Tsujimura, Naoto; Iijima, Shohei; Takachi, Ko; Nakahara, Yujiro et al. (2017-08-24). "CUBIC pathology: three-dimensional imaging for pathological diagnosis". Scientific Reports 7 (1): 9269. doi:10.1038/s41598-017-09117-0. ISSN 2045-2322. PMID 28839164. Bibcode: 2017NatSR...7.9269N. 
  11. ↑ Animal Tissue-Clearing Reagents, TCI
  12. ↑ Yu, Tingting; Qi, Yisong; Gong, Hui; Luo, Qingming; Zhu, Dan (2018). "Optical clearing for multiscale biological tissues". Journal of Biophotonics 11 (2): e201700187. doi:10.1002/jbio.201700187. ISSN 1864-0648. PMID 29024450. 
  13. ↑ Lloyd-Lewis, Bethan; Davis, Felicity M.; Harris, Olivia B.; Hitchcock, Jessica R.; Lourenco, Filipe C.; Pasche, Mathias; Watson, Christine J. (2016-12-13). "Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods". Breast Cancer Research 18 (1): 127. doi:10.1186/s13058-016-0754-9. ISSN 1465-542X. PMID 27964754. 
  14. ↑ 14.0 14.1 Hama, Hiroshi; Hioki, Hiroyuki; Namiki, Kana; Hoshida, Tetsushi; Kurokawa, Hiroshi; Ishidate, Fumiyoshi; Kaneko, Takeshi; Akagi, Takumi et al. (October 2015). "ScaleS: an optical clearing palette for biological imaging". Nature Neuroscience 18 (10): 1518–1529. doi:10.1038/nn.4107. ISSN 1546-1726. PMID 26368944. 
  15. ↑ Gómez-Gaviro, María Victoria; Balaban, Evan; Bocancea, Diana; Lorrio, María Teresa; Pompeiano, Maria; Desco, Manuel; Ripoll, Jorge; Vaquero, Juan José (2017-06-01). "Optimized CUBIC protocol for three-dimensional imaging of chicken embryos at single-cell resolution". Development 144 (11): 2092–2097. doi:10.1242/dev.145805. ISSN 0950-1991. PMID 28432219. https://e-archivo.uc3m.es/bitstream/10016/24702/1/optimized_DEVELOPMENT_2017.pdf. 
  16. ↑ Kubota, Shimpei I.; Takahashi, Kei; Nishida, Jun; Morishita, Yasuyuki; Ehata, Shogo; Tainaka, Kazuki; Miyazono, Kohei; Ueda, Hiroki R. (2017). "Whole-Body Profiling of Cancer Metastasis with Single-Cell Resolution". Cell Reports 20 (1): 236–250. doi:10.1016/j.celrep.2017.06.010. PMID 28683317. 



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