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

This work was generously supported by the National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS) grant 1R01NS086981 and the Conrad N. Hilton Foundation. We thank Dr. Laurent Bentolila and Dr. Matthew Schibler for their invaluable assistance with confocal microscopy. We thank those that have contributed to the CLARITY forum (http://forum.claritytechniques.org). We especially thank Dr. Karl Deisseroth for opening up his lab to teach this fascinating technique. The authors are grateful for the generous support from the Brain Mapping Medical Research Organization, Brain Mapping Support Foundation, Pierson-Lovelace Foundation, The Ahmanson Foundation, Capital Group Companies Charitable Foundation, William M. and Linda R. Dietel Philanthropic Fund, and Northstar Fund. Research reported in this publication was also partially supported by the National Center for Research Resources and by the Office of the Director of the National Institutes of Health under award numbers C06RR012169, C06RR015431, and S10OD011939. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

All experiments were performed in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines. THY1-YFP, PLP-EGFP and PV-TdTomato mice were used for these experiments, but any mice expressing fluorescent proteins can be successfully cleared and imaged.
1. Tissue Preparation
Caution: Paraformaldehyde (PFA) and acrylamide are toxic and irritants. Perform experiments utilizing these reagents in a fume hood and with appropriate personal protective equip...

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Passive clearing of hydrogel embedded tissues (passive CLARITY) is a simple and inexpensive method for clearing large pieces of tissue. This approach does not require dedicated equipment and can easily be performed in a temperature-controlled shaker. Over the span of a few weeks, even large, highly myelinated tissues, such as an entire brain or spinal cord, will become transparent and suitable for microscopy. Even though this report has focused on the clearing of CNS tissues, passive CLARITY can be applied to any tissue....

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.

Visualizing the structural organization of the brain is critical for understanding how morphology and connectivity affect brain function in health and disease. Optical clearing techniques make it possible to image cell populations in 3D in intact tissues, allowing us to study morphology and connectivity cohesively.
Mice that express fluorescent proteins driven by promoters specific for sub-populations of cells permit one to teas...

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.

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

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