Name: optical clearing of the mouse central nervous system using passive clarity
Uploaded: 2016-06-30T13:00:00
Description: Watch this Scientific Journal Video about Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY at JoVE.com

وأيد هذا العمل بسخاء من قبل المعاهد الوطنية لل/ معهد الصحة الوطني للاضطرابات العصبية والسكتة الدماغية (NIH / NINDS) منحة 1R01NS086981 ومؤسسة كونراد هيلتون. نشكر الدكتور لوران Bentolila والدكتور ماثيو Schibler للمساعدة لا تقدر بثمن مع المجهر متحد البؤر. نشكر تلك التي ساهمت في المنتدى ضوح (http://forum.claritytechniques.org). نخص بالشكر الدكتور كارل Deisseroth لفتح مختبره لتعليم هذه التقنية الرائعة. الكتاب ممتنون للدعم السخي من منظمة الدماغ رسم الخرائط الأبحاث الطبية، ومؤسسة الدماغ رسم الخرائط دعم، بيرسون-افليس مؤسسة، مؤسسة Ahmanson، مجموعة شركات رأس المال للأعمال الخيرية، ويليام م وليندا صندوق الخيرية Dietel R.، وصندوق نورث ستار . تم البحث عنها في هذا المنشور أيضا بدعم جزئي من قبل المركز الوطني للبحوث الموارد ومكتب المدير الأمةآل معاهد الصحة تحت أرقام جائزة C06RR012169، C06RR015431، وS10OD011939. المحتوى هو فقط من مسؤولية الكتاب ولا تمثل بالضرورة وجهة النظر الرسمية للمعاهد الوطنية للصحة.

أجريت جميع التجارب وفقا للرعاية الحيوان المؤسسية والمبادئ التوجيهية للجنة الاستخدام (IACUC). استخدمت THY1-YFP، حزب العمال التقدمي-EGFP وPV-TdTomato الفئران لهذه التجارب، ولكن أي الفئران معربا عن البروتينات الفلورية يمكن مسح بنجاح وتصويرها. 1. ...

<ol>
	<li>Micheva, K. D., Smith, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Array+tomography:+a+new+tool+for+imaging+the+molecular+architecture+and+ultrastructure+of+neural+circuits.">Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits.</a> Neuron. 55, 25-36 (2007).</li><li>Kleinfeld, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+automated+histology+in+the+pursuit+of+connectomes.">Large-scale automated histology in the pursuit of connectomes.</a> J Neurosci. 31, 16125-16138 (2011).</li><li>MacKenzie-Graham, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multimodal,+multidimensional+atlas+of+the+C57BL/6J+mouse+brain.">A multimodal, multidimensional atlas of the C57BL/6J mouse brain.</a> J Anat. 204, 93-102 (2004).</li><li>Denk, W., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photon+upmanship:+why+multiphoton+imaging+is+more+than+a+gimmick.">Photon upmanship: why multiphoton imaging is more than a gimmick.</a> Neuron. 18, 351-357 (1997).</li><li>Denk, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+and+functional+imaging+of+neurons+using+2-photon+laser+scanning+microscopy.">Anatomical and functional imaging of neurons using 2-photon laser scanning microscopy.</a> J Neurosci Methods. 54, 151-162 (1994).</li><li>Zipfel, W. R., Williams, R. M., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+magic:+multiphoton+microscopy+in+the+biosciences.">Nonlinear magic: multiphoton microscopy in the biosciences.</a> Nat Biotechnol. 21, 1369-1377 (2003).</li><li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+two-photon+microscopy.">Deep tissue two-photon microscopy.</a> Nat Methods. 2, 932-940 (2005).</li><li>Theer, P., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+fundamental+imaging-depth+limit+in+two-photon+microscopy.">On the fundamental imaging-depth limit in two-photon microscopy.</a> J Opt Soc Am A Opt Image Sci. Vis. 23, 3139-3149 (2006).</li><li>Dodt, H. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultramicroscopy:+three-dimensional+visualization+of+neuronal+networks+in+the+whole+mouse+brain.">Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain.</a> Nat Methods. 4, 331-336 (2007).</li><li>Erturk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+imaging+of+solvent-cleared+organs+using+3DISCO.">Three-dimensional imaging of solvent-cleared organs using 3DISCO.</a> Nat Protoc. 7, 1983-1995 (2012).</li><li>Hama, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scale:+a+chemical+approach+for+fluorescence+imaging+and+reconstruction+of+transparent+mouse+brain.">Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain.</a> Nat Neurosci. 14, 1481-1488 (2011).</li><li>Ke, M. T., Fujimoto, S., Imai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SeeDB:+a+simple+and+morphology-preserving+optical+clearing+agent+for+neuronal+circuit+reconstruction.">SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction.</a> Nat Neurosci. 16, 1154-1161 (2013).</li><li>Susaki, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-brain+imaging+with+single-cell+resolution+using+chemical+cocktails+and+computational+analysis.">Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.</a> Cell. 157, 726-739 (2014).</li><li>Chung, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+molecular+interrogation+of+intact+biological+systems.">Structural and molecular interrogation of intact biological systems.</a> Nature. 497, 332-337 (2013).</li><li>Spence, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bringing+CLARITY+to+gray+matter+atrophy.">Bringing CLARITY to gray matter atrophy.</a> Neuroimage. 101, 625-632 (2014).</li><li>Tomer, R., Ye, L., Hsueh, B., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+CLARITY+for+rapid+and+high-resolution+imaging+of+intact+tissues.">Advanced CLARITY for rapid and high-resolution imaging of intact tissues.</a> Nat Protoc. 9, 1682-1697 (2014).</li><li>Yang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+phenotyping+within+transparent+intact+tissue+through+whole-body+clearing.">Single-cell phenotyping within transparent intact tissue through whole-body clearing.</a> Cell. 158, 945-958 (2014).</li><li>Costantini, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+clearing+agent+for+multi-modal+brain+imaging.">A versatile clearing agent for multi-modal brain imaging.</a> Sci Rep. 5, 9808 (2015).</li><li>Zheng, H., Rinaman, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+CLARITY+for+visualizing+immunofluorescence+labeling+in+the+developing+rat+brain.">Simplified CLARITY for visualizing immunofluorescence labeling in the developing rat brain.</a> Brain Struct Funct. , (2015).</li><li>Parra, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphoton+microscopy+of+cleared+mouse+brain+expressing+YFP.">Multiphoton microscopy of cleared mouse brain expressing YFP.</a> J Vis Exp. (67), e3848 (2012).</li><li>Weinger, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+cellular+dynamics+in+the+mouse+spinal+cord.">Two-photon imaging of cellular dynamics in the mouse spinal cord.</a> J Viz Exp. (96), e52580 (2015).</li><li>Hama, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScaleS:+an+optical+clearing+palette+for+biological+imaging.">ScaleS: an optical clearing palette for biological imaging.</a> Nat Neurosci. 18, 1518-1529 (2015).</li><li>Treweek, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-body+tissue+stabilization+and+selective+extractions+via+tissue-hydrogel+hybrids+for+high-resolution+intact+circuit+mapping+and+phenotyping.">Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping.</a> Nat Protoc. 10, 1860-1896 (2015).</li></ol>

المقاصة سلبية من هيدروجيل جزءا لا يتجزأ من الأنسجة (وضوح السلبي) هي طريقة بسيطة وغير مكلفة لإزالة أجزاء كبيرة من الأنسجة. لا يتطلب هذا النهج معدات مخصصة، ويمكن بسهولة أن يؤديها في شاكر التحكم في درجة حرارته. على مدى غضون بضعة أسابيع، حتى الكبيرة، والأنسجة العصبي للغا?...

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 &#181;m in laser scanning confocal microscopy and to less than 800 &#181;m 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.

<table border="0" cellpadding="0" cellspacing="0" width="750">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10X phosphate buffered saline (PBS)</td>
			<td style="width:212px;">Fisher</td>
			<td style="width:119px;">BP399-1</td>
			<td style="width:203px;">buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">32% paraformaldehyde (PFA)</td>
			<td style="width:212px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15714-5</td>
			<td>perfusion and hydrogel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2,2&#39;-thiodiethanol (TDE)</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:119px;">166782-500G</td>
			<td>refractive index matching solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">40% acrylamide</td>
			<td style="width:212px;">Bio-Rad</td>
			<td style="width:119px;">161-0140</td>
			<td>hydrogel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2% bis-acrylamide</td>
			<td style="width:212px;">Bio-Rad</td>
			<td style="width:119px;">161-0142</td>
			<td>hydrogel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VA-044 initiator</td>
			<td>Wako Pure Chemical Industries, Ltd.</td>
			<td>VA044</td>
			<td>hydrogel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">boric acid</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:119px;">B7901</td>
			<td>clearing buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium dodecyl sulfate (SDS)</td>
			<td style="width:212px;">Fisher</td>
			<td style="width:119px;">BP166-5</td>
			<td>clearing buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium hydroxide</td>
			<td style="width:212px;">Fisher</td>
			<td style="width:119px;">55255-1</td>
			<td>buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium azide</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:119px;">52002-100G</td>
			<td>preservative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">triton x-100</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:119px;">X100-500G</td>
			<td>buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">heparin</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3393-50KU</td>
			<td>perfusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium nitrate</td>
			<td style="width:212px;">Fisher</td>
			<td style="width:119px;">BP360-500</td>
			<td>perfusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DAPI</td>
			<td style="width:212px;">Molecular Probes</td>
			<td style="width:119px;">D1306</td>
			<td>nuclear stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">99.9% isoflurane</td>
			<td style="width:212px;">Phoenix</td>
			<td style="width:119px;">57319-559-06</td>
			<td>anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">hydrocholoric acid</td>
			<td style="width:212px;">Fisher</td>
			<td style="width:119px;">A1445-500</td>
			<td>buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">glass bottom dish</td>
			<td style="width:212px;">Willco</td>
			<td style="width:119px;">HBSB-5040</td>
			<td>Willco dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">reusable adhesive</td>
			<td style="width:212px;">Bostick</td>
			<td style="width:119px;">371351</td>
			<td>Blu-Tack</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">silicon elastomer</td>
			<td style="width:212px;">World Precision Instruments</td>
			<td style="width:119px;">KWIK-CAST</td>
			<td>Kwik-Cast</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">lint-free wipe</td>
			<td style="width:212px;">Kimberly-Clark</td>
			<td style="width:119px;">34120</td>
			<td>KimWipe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mouse brain matrix</td>
			<td style="width:212px;">Roboz</td>
			<td style="width:119px;">SA-2175</td>
			<td>sectioning tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">matrix blades</td>
			<td style="width:212px;">Roboz</td>
			<td style="width:119px;">RS-9887</td>
			<td>sectioning tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">peristaltic pump</td>
			<td style="width:212px;">Cole-Parmer</td>
			<td style="width:119px;">77122-24</td>
			<td>pefusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">laser scanning confocal microscope</td>
			<td>Leica</td>
			<td>SP5</td>
			<td>microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">imaging software</td>
			<td style="width:212px;">Leica</td>
			<td style="width:119px;">LAS AF</td>
			<td>microscopy</td>
		</tr>
	</tbody>
</table>

optical clearing of the mouse central nervous system using passive clarity

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&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

تصور التنظيم الهيكلي للدماغ أمر بالغ الأهمية لفهم كيفية التشكل والاتصال تؤثر على وظيفة الدماغ في الصحة والمرض. تقنيات المقاصة البصرية تجعل من الممكن لسكان الخلية الصورة في 3D في الأنسجة سليمة، مما يسمح لنا لدراسة مورفولوجية والربط متماسك. <p class="jov...

Watch this Scientific Journal Video about Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY at JoVE.com

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

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.

المقاصة البصري للنظام العصبي المركزي الماوس عن طريق وضوح السلبي

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

The overall goal of this protocol is to demonstrate modifications to the original CLARITY protocol, that yield more consistent and cost effective results. This method can help answer key questions in neuroscience, such as 3D morphology, and connectivity in the central nervous system. The main advantage of this technique is that it improves the consistency of optical clearing and microscopy in the mouse central nervous system.In a cost effective and reproduceable manner. Visual demonstration of this method is critical. As the imaging chamber construction is complicated and not readily obvious from written description.Obtain paraformaldehyde fixed brain and spinal cord tissue from mice expressing fluorescent proteins. Hemisect the brain by placing it in a plastic mouse brain matrix and cutting along the midline with a matrix blade. Add each hemisphere and spinal cord to separate 50 milliliter centrifuge tubes.Each containing 40 milliliters of hydrogel solution. Cover the tubes with aluminum foil to protect the fluorophores. And incubate the tubes containing samples and hydrogel at four degrees celsius with gentle shaking for seven days.Following the incubation period discard 25 milliliters of hydrogel leaving the sample and approximately 25 milliliters of hydrogel in the centrifuge tube. Next deoxygenate the sample by removing the cap, placing the centrifuge tube in a desiccator jar, and connecting the jar to a vacuum. Apply the vacuum for at least 10 minutes.Gently shake to dislodge the bubbles of emitted oxygen. After 10 minutes, replace the vacuum in the desiccator jar with 100%nitrogen gas over two to three minutes. Once the pressure equalizes and the top of the desiccator jar can be opened, quickly cap the tube to prevent the reintroduction of oxygen.Next polymerize the hydrogel by placing the tube with sample in a 37 degrees celsius water bath for three hours until polymerization is complete. After three hours the polymerized hydrogel will take on a gel like consistency. Use a spatula to remover the polymerized sample from the centrifuge tube then cut away the excess hydrogel from the sample.Lastly, remove the remaining hydrogel by placing the sample on a lint free wipe, and gently rubbing and rolling the tissue on it, leaving behind only a thin layer of polymerized hydrogel on the sample. To begin the process of lipid removal, transfer the polymerized sample to a clean 50 milliliter centrifuge tube with 45 milliliters of clearing buffer, and incubate at 45 degrees celsius with gentle shaking for seven days. Change the buffer daily during this initial seven day period by pouring off the used clearing buffer into a chemical waste container.And adding 45 milliliters of fresh clearing buffer. Once all lipids are solubilized and the tissue reaches transparency transfer the sample to a new 50 milliliter centrifuge tube filled with 45 milliliters of PBST, and wash four times at 40 degrees celsius with gentle shaking over the next 24 hours, to remove residual SDS. After the final PBST wash, transfer the sample to a clean 15 milliliter centrifuge tube filled with one microgram per milliliter DAPI and 1xPBST.Wrap with foil and incubate at room temperature for 24 hours. The next day wash three times in PBST at room temperature for at least six hours per wash. And then proceed to refractive index matching.To begin this part of the procedure, transfer the sample to a clean 15 milliliter centrifuge tube filled with 2-2 thiodiethanol and 1xPBS at PH 7.5 and incubate as shown on screen. Towards the end of the second incubation, create and imaging chamber by rolling a piece of reusable adhesive into a cylinder with uniform diameter just slightly greater than the thickness of the sample. Lay the cylinder in an open ended circle on a 40 millimeter glass bottom dish.Then use a P1000 pipette tip to create a seal between the reusable adhesive and glass by rolling it around the outer rim of the cylinder. Pipette approximately 100 microliters of 63%TDE onto the glass dish and spread it around to cover the glass surface within the circle of reusable adhesive. Then use a spatula to carefully place the sample in the middle of the circle taking care not to form bubbles.Next, pipette another 100 microliters of 63%TDE directly onto the sample, and immediately place a 40 millimeter circular cover glass over the reusable adhesive. Use the index, middle, and ring finger of both hands to carefully press around the perimeter of the circle until the cover glass makes contact with the sample. Now, slowly bring the glass bottom dish to an upright position resting against a surface, then add 63%TDE with a pipette until the solution reaches the small opening at the top.Use a lint free wipe to dry off the pipette tip so that the solution does not drip on the glass. Lastly, add silicon elastomer to the small opening to enclose the circle and create a closed chamber. Wait five minutes to dry, and then proceed to imaging.Place the imaging chamber with the sample inside on the stage of a laser scanning confocal microscope. Open the imaging software, to turn on the argon excitation laser, click the configuration tab at the top of the program, then the laser icon. Check the box next to argon to power the laser and move the slide scale to 30%Return to the acquire tab at the top.In the beam path settings box, go to the load-save setting box, and select the drop down menu to select the appropriate wavelength channel to emit YFP. In the channel settings box, select the appropriate objectives for imaging by clicking the red hyperlink labeled objective current object. Use objectives with a working distance greater than the thickness of the tissue.And a large numerical aperture to maximize the inplane image resolution, and to minimize optical section thickness. On the left column of drop down menus open the XY resolution window to set the acquisition matrix, called format to 1024x1024 or a value appropriate for the lateral resolution limit of the objective. Set the zoom factor as low as possible.1.7 is used here. In the same window set eighth line average and one frame average parameters by dropping down the individually labeled menus accordingly. Locate the sample using the stage controls.Once a representative field is visible, set the gain to between 760 to 780, and the offset to between 1.0 and 1.5 for YFP. Select tile scan. And then set the upper and lower limits of the Z stack to be acquired.Set the thickness of the optical section based on the objectives optical section resolution limit and then start the acquisition. This image shows YFP positive neurons in the cerebral cortex and hippocampus imaged as 5x magnification and reconstructed in 3D. The scale bar represents one millimeter.This maximum intensity projection of a 10x image stack of the cerebral cortex demonsrates the dendritic aborization of cortical layer five pyramidal neurons. Here the scale bar represents 100 microns. Here THY-1YFP expression is shown in an intact cervical and thoracic spinal cord, imaged at 5x magnification, and reconstructed in 3D.The scale bar represents two millimeters. Finally this maximum intensity projection of a 10x imaged stack of the spinal cord, demonstrates individual axons, scale bars represent 100 microns. If preformed properly this technique can be done in two to four weeks for whole spinal cords, and four to six weeks for hemisected brains.Following this procedure, other methods such as immunohistic chemistry can be preformed in order to answer questions that require more complex biochemical phenotyping. After watching this video you should have a good understanding of how to optically clear and image a mouse central nervous system. Using passive clarity, and laser confocal microscopy.

Research

JoVE Journal

Neuroscience

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

التصوير المتعدد الوسائط من زرع الخلايا الجذعية في الجهاز العصبي المركزي من الفئران

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

انتاج ناقلات Lentiviral للخلايا Transducing من الجهاز العصبي المركزي

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

وضع العلامات واحدة من الخلايا في الجهاز العصبي المركزي<em&gt; ذبابة الفاكهة الدروسوفيلا</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

مباشرة داخل البطين تسليم المخدرات الى القوارض الجهاز العصبي المركزي

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

في الجسم الحي الاستجواب من النظام العصبي المركزي Translatome بواسطة Polyribosome تجزئة

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

في الجسم الحي Optogenetic تحفيز الجهاز العصبي المركزي القوارض

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

رواية السلبي مسح أساليب للإنتاج السريع للشفافية الضوئية في كامل أنسجة الجهاز العصبي المركزي

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

تسجيل الكهربية لنشاط الجهاز العصبي المركزي للطور الثالث Melanogaster المورفولوجية

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

زرع الذرية القشرية الهندسية كيميائياً في عقول الفئران المبكرة بعد الولادة

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

توصيل البراز المضاد للندرة في الجهاز العصبي المركزي للفئران

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central ...