組織透明化在神經(jīng)科學領域中的應用
神經(jīng)科學是一門神秘的科學，也一直是生命科學研究的熱點。如果有一天，我們可以搞明白神經(jīng)細胞是怎么一回事，神經(jīng)細胞之間是如何協(xié)作的，或許我們就能明白意識是如何產(chǎn)生的了？明白了我們?yōu)槭裁匆X？明白了男人和女人的大腦有什么不一樣？為了明確神經(jīng)元的結構和功能連接，需要對整個大腦和脊髓中的神經(jīng)元及突觸進行成像和追蹤。組織透明化技術的發(fā)展使生物樣本變得透明，為生物組織結構的三維可視化研究打開了大門，并為進一步探索人類疾病的復雜性提供了嶄新的平臺，近年來組織透明化技術在生物醫(yī)學研究領域得到了廣泛應用。
BRADKE等利用3DISCO透明化技術和光片顯微鏡成像技術識別、追蹤和評估脊髓損傷后的軸突再生，確定了軸突再生能力及再生軸突軌跡，有助于解釋軸突再生機制，同時定量測定了脊髓損傷后神經(jīng)膠質(zhì)的活動反應。
Imaging large regions of the spinal cord of GFP-M mice
(a) Entire spinal cord of a GFP-M mouse cut into 3- to 4-mm-long segments, cleared and imaged with ultramicroscopy. (b) Drawing of the ultramicroscopy setup showing tissue positioning and the light path. (c) A spinal cord segment (length 4 mm, T12 to L2 spine level) of a GFP-M mouse scanned with ultramicroscopy shown in a horizontal view. (d) Cross-view projection (50-µm thickness) of the indicated region in c. White arrows in c,d mark individual axons in the white matter; red arrows mark cell bodies in the gray matter. (e) Traced white and gray matter boundaries and axon bundles (yellow arrows).
阿爾茲海默癥（AD）是一種起病隱匿的進行性發(fā)展的神經(jīng)系統(tǒng)退行性疾病。臨床上以記憶障礙、失語、失用、失認、視空間技能損壞、執(zhí)行功能障礙以及人格和行為改變等全面性癡呆表現(xiàn)為特征，細胞外β淀粉樣蛋白沉積和細胞內(nèi)tau蛋白磷酸化是AD的主要病理特征。Liebmann等人利用iDISCO技術對阿爾茲海默癥模型小鼠腦進行透明化闡明了β淀粉樣蛋白沉積和小膠質(zhì)細胞及脈管系統(tǒng)之間的空間分布關系，這將對阿爾茲海默癥的科學研究和治療產(chǎn)生重大意義。
Double Labeling of β-Amyloid Plaques and Reactive Microgliosis, Axonal Dystrophy, or Vasculature in Cleared AD Brains with Volume Imaging
（A）confocal image of β-amyloid plaques and cell nuclei double staining in a 17-month-old2xTg AD cleared mouse brain. (B) Confocal optical slices of microglia stained with anti-Iba1 antibody. (C) 3D surface rendering of four isolated β-amyloid plaques and neighboring microglia cells from the AD brain described in (B). (D)Maximum intensity projection of β-amyloid plaques and neuro filament H staining in a10-month-old 2xTgAD cleared mouse cortex(500mmthick). (E)Magnified region from (D) showing labeled axons in absence of β-amyloid plaques. (F) Magnified region from (D) showing dystrophic axons surrounding labeled β-amyloid plaques. (G) Maximum intensity projection of optical section (1mm) from the cortex of an 11-month-old 2xTgAD mouse brain labeled for plaques (anti-b amyloid anti-bodies) and vasculature. (H)Maximum intensity projection of optical section (1mm) from the cortex and hippocampus of an 8-month-old 2xTgAD mouse brain labeled for plaques (Congored) and vasculature.
Chung等人通過CLARITY技術獲得透明而完整的小鼠大腦，利用Thy1–eYFP信號實現(xiàn)了對小鼠大腦神經(jīng)元進行遠距離投射、神經(jīng)回路、細胞關系、亞細胞結構、蛋白質(zhì)復合物、 核酸和神經(jīng)遞質(zhì)的成像。展現(xiàn)了大腦中復雜的精細連接和分子結構。
Intact adult mouse brain imaging
a, Cajal quote before CLARITY. b, Cajal quote after CLARITY: Thy1–eYFP line-H mouse brain after hydrogel–tissue hybridization, ETC and refractive-index matching. c, Fluorescence image of brain depicted in b. d, Dorsal aspect is imaged, then brain is inverted and ventral aspect imaged. e, Three-dimensional rendering of clarified brain imaged；f, Non sectioned mouse brain tissue showing cortex, hippocampus and thalamus；g–l, Optical sections from f showing negligible resolution；m, Cross-section of axons in clarified Thy1–channelrhodopsin2 (ChR2)–eYFP striatum；n, Dendrites and spines of neurons in clarified Thy1–eYFP line-H cortex.
MURAKAMI等人應用CUBIC-X透明并膨大小鼠大腦，在亞細胞水平對整個小鼠大腦進行成像，并繪制出了一張小鼠大腦圖譜。他們采用化學方法標記了大腦中的每個細胞，然后在大腦透明化的同時將其尺寸擴大了十倍，利用精密成像技術對神經(jīng)元進行了三維重建，總計約7200萬個細胞。
Construction of a single-cell-resolution mouse brain atlas (CUBIC-Atlas)
a, Overview of construction of the CUBIC-Atlas. b, The CUBIC-Atlas. Horizontal, sagittal and coronal view of single-plane images (left) and volume-rendered images (right) of the CUBIC-Atlas. c–j, Major anatomical areas in the CUBIC-Atlas. k, Overview of whole-brain cell counting in C57BL/6N 8-week-old male mice. l, Cell numbers in each brain area.
目前組織透明化方法有油性，基于水凝膠和水性的組織透明化方法。其中油性組織透明化方法主要有BABB, 3DISCO, uDISCO，F(xiàn)DISCO和 PEGASOS等，基于水凝膠的透明化方法有CLARITY和SHIELD，水性組織透明化方法有CLearT/CLearT2,SeeDB, FRUIT, Scale和CUBIC等方法。而锘海研發(fā)的透明化試劑盒依托測樣服務中積累到的豐富而寶貴的經(jīng)驗，對各種透明化方法進行測試和比較后對試劑配方進行了精心優(yōu)化，使其在組織熒光保護、樣本形態(tài)維持、降低自發(fā)熒光等方面表現(xiàn)出非常優(yōu)異的性能，是研究神經(jīng)科學的不二之選。
锘海組織透明化試劑盒
參考文獻：
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锘海生命科學為廣大客戶提供專業(yè)的生物組織透明化、免疫染色、平鋪光片顯微鏡3D熒光成像、數(shù)據(jù)分析、數(shù)據(jù)存儲等一站式科研服務，旨在通過快速、多樣化的科研服務為每一位生命科學工作者提供個體化/定制化的解決方案。
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