鼠标中枢神经系统的光透明使用被动CLARITY
Summary
Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.
Abstract
Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one’s ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2′-thiodiethanol (TDE), a previously described refractive index matching solution.
Introduction
The ability to image complete neuroanatomical structures is immensely valuable for understanding the brain in health and disease. Traditionally, 3D imaging has required tissue sectioning to provide the axial resolution and to visualize deep anatomical structures. This approach can produce high-resolution data sets, but requires sophisticated image reconstruction techniques and is very labor intensive. As a result, it has been limited to the imaging of small volumes of tissue1-3. Optical sectioning, on the other hand, is well suited for the creation of high-resolution 3D images of fluorescently labeled tissues. Since optical sectioning is inherently three dimensional, it does not require extensive computation to produce a 3D image volume. However, light scattering and tissue opacity limit the depth at which tissues can be optically sectioned. The depth of imaging is limited to about 150 µm in laser scanning confocal microscopy and to less than 800 µm using two-photon excitation microscopy4-8.
In order to overcome these limitations, several optical clearing techniques have been recently developed and then further refined to permit the deep microscopic imaging of intact tissues. The use of benzyl alcohol and benzyl benzoate (BABB) to render fixed tissues transparent was among one of the earliest approaches9. However, this approach was limited by the quenching of fluorescence in the samples and incomplete clearing of highly myelinated structures10. Refinements of this technique, such as 3D Imaging of Solvent Cleared Organs (3DISCO), have led to very rapid and complete tissue clearing, but still suffer from rapid loss of fluorescent signals, especially yellow fluorescent protein (YFP)10. Water-based clearing solutions, such as Sca/e11 and SeeDB12, preserve fluorescent signals, but do not completely clear highly myelinated tissues. Clear, Unobstructed Brain Imaging Cocktails and Computational analysis (CUBIC) is a promising new optical clearing technology that appears to overcome the limitations of previously developed water-based clearing solutions13. In contrast with other optical clearing techniques, Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY)14 embeds the brain in a porous matrix that provides structural integrity to proteins, nucleic acids and small molecules, while leaving lipids unbound. The lipids are then removed electrophoretically, culminating in an optically cleared brain that can be easily visualized and equally easily probed using commonly available techniques.
Electrophoretic tissue clearing (ETC) is used to remove lipids from hydrogel embedded tissues in CLARITY, however, ETC can be difficult to implement consistently and tissues subject to electrophoresis can exhibit tissue distortion, browning, loss of fluorescence and antibody reactivity. Passive lipid clearing avoids these limitations, but requires increased clearing times15-17. Passive clearing also permits the clearing of large numbers of samples in parallel, since it is not limited by the number of ETC apparatuses available. Decreasing the concentration of paraformaldehyde and excluding bis-acrylamide from the hydrogel have led to great increases in the speed of clearing at the expense of greater tissue expansion16. Less expensive alternatives to the refractive index matching solution used in the original CLARITY technique have been developed17-19. 2,2′-thiodiethanol (TDE) is an inexpensive and rapid brain clearing agent that reverses some of the tissue expansion that occurs during lipid removal18. This report incorporates passive clearing of hydrogel embedded tissue and the use of TDE as a refractive index matching solution to the original CLARITY technique to produce a highly reproducible and inexpensive protocol for the optical clearing of the mouse central nervous system.
Protocol
Representative Results
Discussion
水凝胶包埋组织（无源净度）的被动结算是用于清除大块组织的一个简单和廉价的方法。这种方法不要求专用设备，并且可以很容易地在一个温度控制的振荡器进行。在几个星期的跨度，即使是大的，高度髓组织，例如整个脑或脊髓，将成为透明，适用于显微镜。尽管这份报告集中在CNS组织中的清除，被动净度可应用于任何组织。
的光透明的技术的相对优点和弱点已在文献<s…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作是慷慨神经疾病和中风（NIH / NINDS）资助1R01NS086981和康拉德希尔顿N.基金会健康/国立全国学院的支持。我们感谢洛朗邦托利拉博士和马修SCHIBLER博士及其与激光共聚焦显微镜宝贵的援助。我们感谢那些给CLARITY论坛（http://forum.claritytechniques.org）作出了贡献。我们特别感谢卡尔戴瑟罗特博士打开了他的实验室，教这个迷人的技术。笔者是从脑成像医学研究组织，脑成像支持基金会皮尔森 – 洛夫莱斯基金会，基金会阿曼森，资本集团慈善基金会，威廉·M和琳达R. Dietel慈善基金，以及基金北极星的慷慨支持表示感谢。研究本出版物中报也部分由国家研究资源中心和民族的主任办公室的支持在奖项数量C06RR012169，C06RR015431和S10OD011939卫生研究院人。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。
Materials
| 10X phosphate buffered saline (PBS) | Fisher | BP399-1 | buffers |
| 32% paraformaldehyde (PFA) | Electron Microscopy Sciences | 15714-5 | perfusion and hydrogel |
| 2,2'-thiodiethanol (TDE) | Sigma-Aldrich | 166782-500G | refractive index matching solution |
| 40% acrylamide | Bio-Rad | 161-0140 | hydrogel |
| 2% bis-acrylamide | Bio-Rad | 161-0142 | hydrogel |
| VA-044 initiator | Wako Pure Chemical Industries, Ltd. | VA044 | hydrogel |
| boric acid | Sigma-Aldrich | B7901 | clearing buffer |
| sodium dodecyl sulfate (SDS) | Fisher | BP166-5 | clearing buffer |
| sodium hydroxide | Fisher | 55255-1 | buffers |
| sodium azide | Sigma-Aldrich | 52002-100G | preservative |
| triton x-100 | Sigma-Aldrich | X100-500G | buffers |
| heparin | Sigma-Aldrich | H3393-50KU | perfusion |
| sodium nitrate | Fisher | BP360-500 | perfusion |
| DAPI | Molecular Probes | D1306 | nuclear stain |
| 99.9% isoflurane | Phoenix | 57319-559-06 | anesthetic |
| hydrocholoric acid | Fisher | A1445-500 | buffers |
| glass bottom dish | Willco | HBSB-5040 | Willco dishes |
| reusable adhesive | Bostick | 371351 | Blu-Tack |
| silicon elastomer | World Precision Instruments | KWIK-CAST | Kwik-Cast |
| lint-free wipe | Kimberly-Clark | 34120 | KimWipe |
| mouse brain matrix | Roboz | SA-2175 | sectioning tissue |
| matrix blades | Roboz | RS-9887 | sectioning tissue |
| peristaltic pump | Cole-Parmer | 77122-24 | pefusion |
| laser scanning confocal microscope | Leica | SP5 | microscopy |
| imaging software | Leica | LAS AF | microscopy |
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