文章來源于锘海生命科學(xué) ，已獲授權(quán)轉(zhuǎn)載。

神經(jīng)科學(xué)是一門神秘的科學(xué)，也一直是生命科學(xué)研究的熱點(diǎn)。如果有一天，我們可以搞明白神經(jīng)細(xì)胞是怎么一回事，神經(jīng)細(xì)胞之間是如何協(xié)作的，或許我們就能明白意識(shí)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(jié)構(gòu)和功能連接，需要對(duì)整個(gè)大腦和脊髓中的神經(jīng)元及突觸進(jìn)行成像和追蹤。組織透明化技術(shù)的發(fā)展使生物樣本變得透明，為生物組織結(jié)構(gòu)的三維可視化研究打開了大門，并為進(jìn)一步探索人類疾病的復(fù)雜性提供了嶄新的平臺(tái)，近年來組織透明化技術(shù)在生物醫(yī)學(xué)研究領(lǐng)域得到了廣泛應(yīng)用。

BRADKE等利用3DISCO透明化技術(shù)和光片顯微鏡成像技術(shù)識(shí)別、追蹤和評(píng)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(jī)制，同時(shí)定量測(cè)定了脊髓損傷后神經(jīng)膠質(zhì)的活動(dòng)反應(yīng)。

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(jìn)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(rèn)、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(xì)胞外β淀粉樣蛋白沉積和細(xì)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(shù)對(duì)阿爾茲海默癥模型小鼠腦進(jìn)行透明化闡明了β淀粉樣蛋白沉積和小膠質(zhì)細(xì)胞及脈管系統(tǒng)之間的空間分布關(guān)系，這將對(duì)阿爾茲海默癥的科學(xué)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(shù)獲得透明而完整的小鼠大腦，利用Thy1–eYFP信號(hào)實(shí)現(xiàn)了對(duì)小鼠大腦神經(jīng)元進(jìn)行遠(yuǎn)距離投射、神經(jīng)回路、細(xì)胞關(guān)系、亞細(xì)胞結(jié)構(gòu)、蛋白質(zhì)復(fù)合物、 核酸和神經(jīng)遞質(zhì)的成像。展現(xiàn)了大腦中復(fù)雜的精細(xì)連接和分子結(jié)構(gòu)。

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(yīng)用CUBIC-X透明并膨大小鼠大腦，在亞細(xì)胞水平對(duì)整個(gè)小鼠大腦進(jìn)行成像，并繪制出了一張小鼠大腦圖譜。他們采用化學(xué)方法標(biāo)記了大腦中的每個(gè)細(xì)胞，然后在大腦透明化的同時(shí)將其尺寸擴(kuò)大了十倍，利用精密成像技術(shù)對(duì)神經(jīng)元進(jìn)行了三維重建，總計(jì)約7200萬個(gè)細(xì)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.

總之，組織透明技術(shù)結(jié)合顯微成像技術(shù)，極大推動(dòng)了全身或全器官成像的進(jìn)程，有助于加強(qiáng)對(duì)完整生物系統(tǒng)的綜合理解，為神經(jīng)科學(xué)研究提供了更加細(xì)致的信息，是神經(jīng)科學(xué)研究領(lǐng)域強(qiáng)有利的工具。

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。而锘海最新研發(fā)的透明化試劑盒依托測(cè)樣服務(wù)中積累到的豐富而寶貴的經(jīng)驗(yàn)，對(duì)各種透明化方法進(jìn)行測(cè)試和比較后對(duì)試劑配方進(jìn)行了精心優(yōu)化，使其在組織熒光保護(hù)、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(xué)的不二之選。

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