Clearing ottica del sistema nervoso centrale del mouse Utilizzando CHIAREZZA passivo
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
compensazione passiva di idrogel incorporato tessuti (chiarezza passivo) è un metodo semplice e poco costoso per la compensazione grandi pezzi di tessuto. Questo approccio non richiede attrezzature dedicate e può essere facilmente eseguita con un agitatore a temperatura controllata. Nell'arco di poche settimane, anche di grandi dimensioni, i tessuti altamente mieliniche, come ad esempio un intero cervello o del midollo spinale, diventeranno trasparenti e adatto per la microscopia. Anche se questo rapporto è conce…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Questo lavoro è stato generosamente sostenuto dal National Institutes of Health / Istituto Nazionale di Malattie Neurologiche e Stroke (/ NINDS NIH) concessione 1R01NS086981 e l'Hilton Fondazione Conrad N.. Ringraziamo il Dr. Laurent Bentolila e il Dr. Matthew Schibler per la preziosa assistenza con microscopia confocale. Ringraziamo coloro che hanno contribuito al forum chiarezza (http://forum.claritytechniques.org). Ringraziamo in modo particolare il Dr. Karl Deisseroth per aprire il suo laboratorio per insegnare questa affascinante tecnica. Gli autori sono grati per il generoso sostegno da parte della Organizzazione Brain Mapping Medical Research, Cervello Fondazione supporta il mapping, Pierson-Lovelace Foundation, la Fondazione Ahmanson, Capital Group Aziende Charitable Foundation, William M. e Linda R. Dietel Fondo filantropico, e il Fondo Northstar . La ricerca riportato nella presente pubblicazione è anche parzialmente sostenuto dal Centro nazionale per le risorse di ricerca e dall'Ufficio del direttore della NazioneAl Institutes of Health ai numeri premio C06RR012169, C06RR015431, e S10OD011939. Il contenuto è di esclusiva responsabilità degli autori e non rappresentano necessariamente il punto di vista ufficiale del National Institutes of Health.
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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