パッシブCLARITYを使用したマウス中枢神経系の光学クリア
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
ハイドロゲル包埋組織（パッシブCLARITY）の受動クリアは組織の大部分をクリアするための簡単で安価な方法です。このアプローチは、専用の設備を必要とせず、容易に温度制御シェーカー中で行うことができます。数週間のスパンで、このような脳全体または脊髄などであっても大規模な、非常に有髄組織は、顕微鏡検査のために透明性と適切ななります。このレポートは、CNS組織の清…
Declarações
The authors have nothing to disclose.
Acknowledgements
この作品は、寛大に神経疾患・脳卒中（NIH / NINDS）助成金1R01NS086981とコンラッドN.ヒルトン財団の健康/国立研究所の国立研究所によってサポートされていました。我々は、共焦点顕微鏡との貴重な援助のために博士ローランBentolila博士マシューSchiblerに感謝します。私たちは、CLARITYフォーラム（http://forum.claritytechniques.org）に寄与しているものに感謝します。我々は、特にこの魅力的な技術を教えるために、彼の研究室を開放するための博士カールDeisserothに感謝します。著者らは、脳マッピング医学研究機構、脳マッピング支援財団、ピアソン・ラブレス財団、アーマンソン財団、キャピタル・グループ会社慈善財団、ウィリアムM.とリンダR. Dietel慈善基金、およびノーススター基金からの寛大な支援に感謝してい。この刊行物で報告された研究はまた、部分的に研究資源のための国立センターと国家のディレクターのオフィスでサポートされていました受賞番号C06RR012169、C06RR015431、およびS10OD011939下の健康のアル研究所。内容はもっぱら著者の責任であり、必ずしも国立衛生研究所の公式見解を示すものではありません。
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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