Name: imaging cleared intact biological systems at a cellular level by 3disco
Uploaded: 2014-07-07T14:00:00
Duration: 7 min 49 s
Description: Watch this Scientific Journal Video about Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO at JoVE.com

We thank C. Hojer for critical reading of the manuscript, B. Bingol (Genentech) for participation in script narrations, N. Bien-Ly for sharing the experience of improved vasculature imaging with tail vein injection of Lectin dye, and M Solloway (Genentech) for the mice used in pancreas imaging. GFP-M line was created by J. Sanes (Washington University in St. Louis) and CX3CR1-GFP mice by R. Littman (NYU). The work is supported by Genentech, Inc.

All animal experiments were performed in accordance with IACUC (Institutional Animal Care and Use Committee) regulations on mice ~3-5 months old. The author declares no competing financial interests.
1. Animal Perfusion and Tissue Preparation
Timing: 30-60 min per mouse + post-fixation (a few hours to overnight).
<ol>
	<li>Weigh the animal and anesthetize using ketamine (80-200 mg/kg) and xylazine (7-20 mg/kg) or 2.5% avertin (0.5 ml/25 g body weight IP).</li>
	<li>Wait a few minutes for anesthesia to take complete effect.</li>
	<li>Pinch the toe and tail of the animal to make sure that the animal is fully anesthetized.</li>
	<li>Perfuse the animal first at RT with 0.1 M Phosphate Buffer (PB) or 0.1 M Phosphate Buffer Saline (PBS) for 5-10 min until the blood is completely removed from the tissue.</li>
	<li>Switch the perfusion to fixative solution: 4% PFA in 0.1 M PB (or 0.1 M PBS) and continue perfusion with 4% PFA for 30-40 min at a speed of 3 ml/min.</li>
	<li>Dissect the organ/s of interest carefully without damaging, e.g., avoid puncturing and squeezing the tissue that is being dissected.</li>
	<li>Remove the extra tissue surrounding the organs (connective tissue, meninges or dura matter) in a petri dish filled with PBS.</li>
	<li>Post-fix the organs in 4% PFA for a few hr or overnight&#160;at 4 &#186;C. Avoid long post-fixation because PFA might quench the signal or increase the autofluorecense overtime10 especially for GFP channel (which is a lesser problem in red and far red channels).</li>
	<li>Wash the organs 2-3x&#160;with PBS at RT just before starting the clearing procedure.</li>
</ol>
2. Tissue Clearing
Timing: 2-3 hr for small organs and 1-4 days for large organs.
Notes: The fluorescent labeling of the tissues by transgene expression, viral transfection, dye tracing or antibody labeling should be completed before clearing. All tissue clearing steps are performed at RT. The clearing solutions THF, DBE, BABB (1 part benzyl alcohol and 2 part benzyl benzoate) and dichloromethane are toxic and inhalation (conduct experiments in properly ventilated fume hood) and direct contact with skin should be avoided (use nitrile gloves).
<ol>
	<li>Prepare 50% (vol/vol), 70%, and 80% THF dilutions in distilled water in separate glass bottles as follows: For 50% THF, e.g., mix 25 ml THF and 25 ml of distilled water in a glass bottle with volume of larger than 50 ml.</li>
	<li>Mix solutions by gently shaking the bottle for a 1-2 min.</li>
	<li>Label the glass bottle clearly.</li>
	<li>Securely close the bottles and keep the working solutions in a dark cabinet. For the best results, do not use the same working solutions longer than 1-2 weeks. Therefore, order the clearing solutions as the smallest volume available to be able use fresh stock reagents.</li>
	<li>Fill the glass vials (e.g., 2-3 ml) with the first clearing solution, 50% THF, and transfer the organs from PBS into glass vials to start clearing.</li>
	<li>Securely close the glass vials with their lids and use a rotator (e.g., a wheel stirrer) for stirring.</li>
	<li>Use aluminum foil to cover and keep the glass vials in dark. Start the rotator to stir the samples in the glass vials for the indicated time (Table 1) at a constant speed (~30 rpm).</li>
	<li>Remove the clearing solution and add the next one in the protocol when the time in Table 1 is completed.</li>
	<li>Collect the clearing waste into glass waste containers that are kept in a hood. 
	Repeat previous step for each clearing solution in the protocol until the end. Use a new pasteur pipette when a new clearing solution is added (e.g., changing from THF to DBE).</li>
	<li>At the final clearing step with BABB or DBE, the incubation times can be extended or shortened until the samples become completely transparent in visible light (or yellowish/transparent if it is a very large tissue like brain).</li>
	<li>Keep the cleared organs in the final clearing solution at all times including the imaging steps.</li>
</ol>
3. Preparing Cleared Organs for Imaging
Timing: 5-15 min.
Note: Image the cleared organs as soon as a complete clearing is achieved. To this end, follow the next steps to prepare the samples for light-sheet microscopy (e.g., ultramicroscopy) or confocal/multi-photon microscopy imaging.
Note: Cleared organs will loose their fluorescence strength over time (particularly fluorescent proteins e.g., GFP; antibody labeling is more resistant and may even give better results with longer incubations). For imaging, various fluorescent microscopy techniques can be used as long as proper handling of the organs in the clearing solution is achieved.
<ol>
	<li>For light-sheet microscopy
	<ol>
		<li>Place or mount the sample appropriately.</li>
		<li>Fix the cleared organ by manually turning the screw of the sample holder4.</li>
		<li>Dip the sample into the imaging chamber (preferably made of glass) of thelight-sheet microscope that is filled with BABB or DBE, whichever was used at the final clearing step.</li>
	</ol>
	</li>
	<li>For multi photon/confocal microscopy
	<ol>
		<li>Mount the sample on an imaging slide (or an imaging chamber) with the final imaging solution to be able to image with a multi photon or confocal microscopy, which typically use oil or water immersed objectives.</li>
		<li>Use dental cement to make a border around the tissue.</li>
		<li>Fill the pool with BABB or DBE just before dental cement gets fully rigid. 
		Note: The dental cement solidifies quickly; practice a few times to get familiar with it before trying the actual samples.</li>
		<li>Immediately place the cleared sample in the middle of the pool and cover it with a cover glass.</li>
		<li>Press the cover glass until it is completely sealed by dental cement and touches at the surface of the cleared organ, which will allow reaching the maximum imaging depth by confocal/multi-photon microscopy. 
		Notes: This ensures that no clearing solution will be spilled during the imaging. Avoid dipping the imaging lens directly into clearing solution, which can harm the lens unless it is resistant to BABB/DBE or has a protective cover.</li>
	</ol>
	</li>
</ol>
4. Imaging Cleared Organs
Timing: 15-45 min.
<ol>
	<li>Collect a z-scan covering the entire cleared tissue (if the used lens allows) at the best resolution that the microscope can deliver.</li>
	<li>Zoom in on the regions of interest to collect higher resolution images.</li>
</ol>
5. Examination of the Data with Software Amira
<ol>
	<li>Load the image series to software Amira with ResolveRT module.</li>
	<li>Enter the correct voxel sizes in &#8220;Image Read Parameter&#8221; window and press ok.</li>
	<li>Select the &#8220;Multi Planer View&#8221; sub-application. Then use the &#8220;Thickness&#8221; slider to choose the optimum threshold for the gray values, adjusting the 2D and 3D values.</li>
	<li>Visualize the sample by browsing the slices in different 2D orientations and using crop-corner module in 3D view. 
	Note: A detailed troubleshooting guide of the protocol can be found in Ert&#252;rk et al4.</li>
</ol>

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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+the+unsectioned+adult+spinal+cord+to+assess+axon+regeneration+and+glial+responses+after+injury.">Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury.</a> Nat Med. 18, 166-171 (2012).</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>Becker, K., Jahrling, N., Saghafi, S., Weiler, R., Dodt, H. 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There is nothing to disclose.

Tissue clearing methods aim to reduce scattering of imaging light by matching the refractive indices of different tissue layers in the cleared organs. As a result, the imaging light can penetrate deep into tissues and stimulate the labeled cells/molecules. This old concept of tissue clearing17 has gained growing attention after the first sophisticated application of the technique on intact mouse brains by Dodt and colleagues2. Since then, there is a rapidly growing list of new clearing methods including 3DISCO, Sca/e, ClearT2, CLARITY and SeeDB3,4,7,18-20. In essence, they all aim to render the organs transparent for deep tissue imaging by preserving the fluorescent label. Among them, 3DISCO stands out as one of the most straightforward and reproducible methods. It is relatively fast compared to other clearing methods mentioned above (1-5 days vs. 2-3 weeks for adult brains, respectively)21. It has already been successfully applied to different organs such as the brain, spinal cord, lymph nodes, spleen, lung, solid tumors, pancreas, and mammary glands3,4,9. In addition, several publications by independent research groups already used this method to obtain imaging results22,23. Nevertheless, different tissue clearing procedures could be advantageous for different conditions. For instance, while Sca/e and SeeDB are applicable best on embryonic tissues6,7, they are water-based clearing methods, which could be easier to handle during imaging. In contrast, CLARITY is a complicated and costly clearing method. But it can be advantageous in the context of antibody labeling, especially in large tissues such as brain8.
The most critical step to obtain the best imaging results from 3DISCO is tissue labeling, which is accomplished before the tissue clearing. It is essential to start with the strongest fluorescent signal possible. This can be achieved in various ways including transgenic expression of fluorescent proteins, tracing by synthetic dyes, viral transfection or antibody labeling. It should be kept in mind that any clearing procedure will reduce the fluorescent signal of the tissues. Hence, if the fluorescent signal is not strong enough at the beginning &#8722;i.e. it is not readily detectable before the clearing&#8722; imaging of the cleared tissues will deliver poor results.
There are some limitations of clearing methods that are waiting for improvements. A critical limitation is that since clearing reagents alter the structure of the cleared organs, they cannot be used on live tissue. Hence, experiments such as with photoactivatable mEos224 should be performed before fixing and clearing. Upon clearing, super-resolution imaging of the fluorescent signal from bright and photostable proteins becomes possible. In addition, because the clearing protocol diminishes the intensity of the fluorescent proteins, the cleared organs cannot be stored for prolonged periods. It is important to note that some organs are harder to be cleared. Especially, if the dissected organs retain high amounts of blood cells (e.g., spleen, liver), they are relatively difficult to clear and image. It is also relatively harder to achieve a complete clearing for large tissues, such as adult rodent brain. Such tissues may need longer incubation times, which could, on the other hand, reduce the fluorescent signal. In addition, development of microscopy methods that can image large transparent organs at once at a high-resolution is an awaiting need to be addressed. Another important limitation of the technique is tissue labeling. While a strong fluorescent signal via genetic or virus expression is ideal, not every cell or molecule can be labeled genetically or virally. On the other hand, antibody labeling of thick tissues is not a straightforward procedure or even not possible in most cases. It may need special permeabilization protocols and long incubation times (several days to a few weeks)3. It is also important to keep in mind that the tissues shrink ~20% in each dimension (measured for spinal cord tissue) after clearing mainly due to dehydration and delipidation. This shrinkage occurs ratiometrically and should be corrected in quantification steps. As observed in the presented results, fluorescently labeled structures remain intact, which is the indication of protein integrity in the cleared tissues. Because of the hydrophobic nature of the final cleared tissue, it cannot be further stained with antibodies or embedded in water containing solutions e.g., Focusclear&#8211;which is used in CLARITY&#8211; or 80% glycerol. Finally, it is a challenge to analyze overwhelming sizes of imaging data. For example, when an entire murine brain is scanned at a cellular resolution, it would be very difficult to trace and quantify desired structures (e.g., neuronal or glia networks) and compare over different samples in an automated way. In summary, while researchers aim to find out new clearing methods, the various challenges above (difficulty of clearing diverse tissues, imaging at high resolution of larger areas, and complicated data analysis) should be improved in parallel.
In conclusion, recently developed tissue clearing techniques including 3DISCO, elegantly demonstrate that high-resolution 3D imaging of intact organs is now possible. Using these methods, scientists can obtain anatomical information of cells and molecules in the intact organ. These pioneering 3D imaging studies on transparent organs inspire a growing number of researchers to decipher complex anatomical and molecular organizations of tissues/organs in heath and disease.

The histological examination of biological tissues is a fundamental approach in understanding the nature of molecules/cells in their natural context. Ideally, high-resolution imaging of the entire organ is most informative and desired. Because light has a limited penetration depth, due to scattering1, fixed organs have to be sectioned in order to perform high-resolution microscopic imaging. Hence, most of the histology studies are performed on few tissue sections representing only a small portion of the organ. In some studies,&#160;e.g., those aiming to trace out neuronal connections in the brain or spinal cord,&#160;all tissue sections from the target organs are collected and imaged for a 3D reconstruction. However, tissue sectioning and subsequent imaging of individual sections has various limitations. These include being time consuming and leading to an incomplete 3D reconstruction of the tissue, due to mechanical distortions and difficulties in the alignment of the resulting images.
Recently, clearing and imaging intact transparent organs has been developed as a significant solution to this shortcoming2,3. Upon clearing, the entire organ is rendered transparent allowing the imaging light to travel end-to-end (Figure 1) to produce high-resolution images of the unsectioned organ using a laser scanning microscope such as a multi photon or light-sheet microscope (Figure 2). Various research groups have developed new tissue clearing protocols to be able to image their tissue of interest for different purposes. These include organic solvent2-5, water6,7 and electrophoresis based8 clearing protocols. Among them, 3-dimensional imaging of solvent cleared organs or 3DISCO is a readily applicable protocol on a variety of biological samples including central nervous system (CNS) organs, immune organs and solid tumors. In addition, it can be combined with different microscopy techniques such as light sheet fluorescence microscopy (LSFM), multi photon and confocal laser scanning microscopy. 3DISCO is based on clearing with readily available and inexpensive reagents such as tetrahydrofuran (THF) and dibenzyl ether (DBE)4. The entire protocol can take as short as 3-4 hr. Thus, 3DISCO is a robust and fast technique compared to traditional histological methods that may take weeks to months to complete9.

<table border="0" cellpadding="0" cellspacing="0" width="913">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:201px;">Thy-1 GFP (GFP-M) mouse</td>
			<td style="width:248px;"></td>
			<td style="width:175px;"></td>
			<td style="width:291px;">can be obtained from Jackson&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CX3CR1 GFP mouse</td>
			<td></td>
			<td></td>
			<td>can be obtained from Littman lab at NYU</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TgN(hGFAP-ECFP) mouse</td>
			<td></td>
			<td></td>
			<td>can be obtained from Jackson&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde&#160;</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Na2HPO4&#160;</td>
			<td>Mallinckrodt</td>
			<td>7917-16</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">NaH2PO4&#160;</td>
			<td>(Mallinckrodt, 7892-04)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-2-2 Tribromoethanol</td>
			<td>Sigma</td>
			<td>T48402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Methyl-2-butanol</td>
			<td>Sigma</td>
			<td align="right">152463</td>
			<td>used to prepare Avertin solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline&#160;</td>
			<td>Braun</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetrahydrofuran&#160;</td>
			<td>Sigma</td>
			<td>186562-12X100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dichloromethane&#160;</td>
			<td>Sigma</td>
			<td>270997-12X100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dibenzyl ether&#160;</td>
			<td>Sigma</td>
			<td>Sigma, 108014-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl alcohol&#160;</td>
			<td>Sigma</td>
			<td>305197-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl benzoate&#160;</td>
			<td>Sigma</td>
			<td>B6630-250ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lectin FITC&#160;</td>
			<td>Sigma</td>
			<td>L0401-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-CC10</td>
			<td>Santa Cruz</td>
			<td>SC-9772</td>
			<td align="right">0.388888889</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin&#160;</td>
			<td>APP pharmaceuticals&#160;</td>
			<td align="right">504001</td>
			<td>used in perfusion&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass vials&#160;</td>
			<td>VWR</td>
			<td>548-0554</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps&#160;</td>
			<td>FST</td>
			<td>11251-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini Rotator&#160;</td>
			<td>VWR</td>
			<td>100498-878</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminum Foil&#160;</td>
			<td>Fisherbrand</td>
			<td>01-213-104</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">100mL Media Storage Bottles</td>
			<td>Kimax Media Bottles</td>
			<td>EW-34523-00</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Minipuls evolution perfusion pump&#160; with MF4* medium flow pump head&#160;</td>
			<td>Gilson, Inc</td>
			<td>F110701 and F117606</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscopy slide</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastWell</td>
			<td>Grace Bio-Labs</td>
			<td>FW20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover slip</td>
			<td>Corning</td>
			<td>2940-245</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass petri dish&#160;</td>
			<td>Corning Pyrex</td>
			<td>3160-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dental cement&#160;</td>
			<td>Lang Dental</td>
			<td>1223PNK</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Quantofix Peroxide Test Strips</td>
			<td>Sigma</td>
			<td align="right">37206</td>
			<td></td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:201px;">Amira with RT resolve microscopy module 
			&#160;</td>
			<td>Visage Imaging</td>
			<td>image analysis software</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:201px;">Imaris</td>
			<td>Bitplane imaging</td>
			<td>image analysis software</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ&#160;</td>
			<td>NIH</td>
			<td>freeware</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging cleared intact biological systems at a cellular level by 3disco

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact organs. Unlike traditional histological methods, where the tissue of interest is sectioned for fluorescent imaging, 3D imaging of cleared tissue allows examination of labeled cells and molecules in the entire specimen. To this end, optically opaque tissues should be rendered transparent by matching the refractory indices throughout the tissue. Subsequently, the tissue can be imaged at once using laser-scanning microscopes to obtain a complete high-resolution 3D image of the specimen. A growing list of tissue clearing protocols including 3DISCO, CLARITY, Sca/e, ClearT2, and SeeDB provide new ways for researchers to image their tissue of interest as a whole. Among them, 3DISCO is a highly reproducible and straightforward method, which can clear different types of tissues and can be utilized with various microscopy techniques. This protocol describes this straightforward procedure and presents its various applications. It also discusses the limitations and possible difficulties and how to overcome them.

Neurons are highly polarized cells with extremely long morphology. Their function depends on the connections that they form between each other and with other tissues. Hence, mapping the structural organization of the neurons is extremely important in order to understand how they function in health and disease. However, tracing such enormous neuronal connections throughout the entire nervous system &#8211;recently named connectomics11&#8722; still remains one of the most difficult challenges in neuroscience. To this end, both EU12 and US13 have initiated large projects to map the human brain.
While 3DISCO can be employed on various organs, it has been particularly useful to trace long neuronal connections in the spinal cord and brain. For example, using ultramicroscopy scans of large cleared spinal cord segments, the axonal connections can be followed over centimeters in the rodent spinal cord (Figure 2). In a similar way, the entire cleared mouse brain (Figure 3a) and hippocampus (Figure 3b) can be imaged to follow neuronal connections in the brain.
When confocal or multi photon microscopy is used on cleared organs, the imaging resolution can be significantly improved, especially in z-dimension. For example, multi photon imaging of cleared spinal cords from GFP-M line mice (Figure 4a) achieves a seamless image throughout the entire depth (~1.5-2 mm) of the spinal cord. Confocal microcopy on the cleared spinal cord delivers improved resolution in a similar way (Figures 4b and c). Multi photon microscopy imaging of cleared brains delivers very high-resolution images to visualize fine details of neuronal structures including dendritic spines (Figure 5). The samples for 3DISCO can be labeled in various ways including transgene expression, viral transfection, dye tracing and antibody labeling. For example, it is possible to label the entire vasculature of the brain (Figures 6a and b) and spinal cord (Figures 6c and d) using lectin conjugated fluorescent tracers4, which can be used to study the blood-brain-barrier (BBB) in health and disease.
Both microglia and astrocytes are highly implicated in the pathology of neurodegeneration including Alzheimer&#8217;s diseases and traumatic injuries14,15. Using 3DISCO, their density and distribution in the spinal cord (Figure 7) or brain can be studied.
Non-neuronal tissues can also be imaged. For example, Clara cells in the entire rodent lungs can be immunolabeled with antibodies and imaged without sectioning at a single cell level (Figure 8). Similarly, it is possible to clear and image cells e.g. alpha cells in the unsectioned pancreas tissue (Figure 9).
<img alt="Figure 1" src="/files/ftp_upload/51382/51382fig1v2highres.jpg" /> 
Figure 1. 3DISCO tissue clearing renders unsectioned tissues transparent for deep tissue imaging. Uncleared (a) and cleared (b) spinal cord tissues as seen by visible light. Upon clearing, deep tissue laser-scanning microscopy becomes possible. (c) Uncleared and cleared spinal cord tissues were imaged by 2-photon microscopy. Scale bars in a, b = 0.5 mm and in c = 100 &#181;m.
<img alt="Figure 2" src="/files/ftp_upload/51382/51382fig2v2highres.jpg" /> 
Figure 2. 3DISCO imaging of spinal cord to follow axonal extensions. The dissected spinal cord from Thy-1 GFP transgenic mouse line (GFP-M) is divided into smaller pieces (~4 mm). After following clearing protocol for small tissues (Table 1) the transparent spinal cords were visualized using ultramicroscopy. 3D reconstructions of a ~4 mm spinal cord segment in horizontal (a), coronal (b) and sagittal view (c). (d) Representative traced axons (red) are shown in the grayscale transparent view. (e) High magnification view of the indicated region in (d). Scale bars in a, b, c, d = 0.5 mm and in e = 20 &#181;m.
<img alt="Figure 3" src="/files/ftp_upload/51382/51382fig3v2highres.jpg" /> 
Figure 3. 3DISCO imaging of cleared brain and hippocampus. Examples of cleared brains and hippocampi of GFP-M mice were imaged with an ultramicroscope. 3D visualizations of the entire brain (a) and hippocampus (b) demonstrating the neuronal networks in the imaged transparent tissues. Scale bars in a = 2 mm and in b = 20 &#181;m.
<img alt="Figure 4" src="/files/ftp_upload/51382/51382fig4v2highres.jpg" /> 
Figure 4. Tissue clearing enhances the resolution obtained by multi photon and confocal microscopy. Transparent spinal cord segments from GFP-M mice imaged by multi photon (a) or confocal microscopy (b, c). Note that clearing substantially improves the resolution and imaging depth revealing the fine structure of axons and dendrites. Scale bars in a = 100 &#181;m and in b, c = 20 &#181;m.
<img alt="Figure 5" src="/files/ftp_upload/51382/51382fig5v2highres.jpg" /> 
Figure 5. 3DISCO imaging of dendritic spines. Transparent brain tissue from GFP-M mice imaged by multi photon. 3D visualizations of scanned cortex region presented in vertical (a) and horizontal perspectives (b). (c) ~50 &#181;m projection from the indicated levels in (a, b). (d) High magnification of the indicated region in (c) demonstrating the fine details of the neuronal structures including dendritic spines (arrowheads). Scale bars in a = 50 &#181;m, in b, c = 25 &#181;m, in d = 5 &#181;m.
<img alt="Figure 6" src="/files/ftp_upload/51382/51382fig6v2highres.jpg" /> 
Figure 6. 3DISCO of vasculature. To label the blood vessels in the entire organs, lectin-FITC dye was used during the perfusion (before the clearing) as described16. It is noteworthy to mention that we found that tail vein injection of the tracer gives better signal over cardiac perfusion of the tracer. After collecting the fixed brains and spinal cords, they were cleared and imaged by ultramicroscopy. 3D visualization of the entire mouse brain vasculature in heatmap (a) and gray-scale (a). (b) 3D visualization of the vasculature of a mouse spinal cord segment in heatmap (c) and gray-scale (d). The heatmaps were generated based on the intensity of lectin staining; blue: low intensity (background) and red: high intensity (vasculature). Scale bars in a = 2 mm, b = 1 mm, and in c, d = 250 &#181;m.
<img alt="Figure 7" src="/files/ftp_upload/51382/51382fig7v2highres.jpg" /> 
Figure 7. 3DISCO of glia cells in cleared CNS tissue. The spinal cords of transgenic mice expressing GFP in microglia TgH(CX3CR1-EGFP) or astrocytes TgN(hGFAP-ECFP) were cleared and imaged by multi photon microscopy. 3D rendering of microglia is shown as surface volume visualization (a) and transparent view (b). (c) An optical projection (~50 &#181;m) is presented to show the details of microglia in the spinal cord. 3D rendering of astrocytes is shown as surface volume visualization (a) and transparent view (b). (f) An optical projection (~50 &#181;m) is presented to show the details of the astrocytes in the spinal cord. Scale bars in a, b, d, e = 100 &#181;m and in c, f = 50 &#181;m.
<img alt="Figure 8" src="/files/ftp_upload/51382/51382fig8v2highres.jpg" width="500" /> 
Figure 8. 3DISCO of a whole-mount lung lobe with antibody staining of clara cells. For the whole-mount antibody staining of lung lobes, 10-week-old BALB/C female mice were perfused through the right ventricle with PBS in order to remove blood from the lungs. Lungs were subsequently inflated with 4% PFA and fixed O/N at RT in fixative at least three times the volume of the tissue. Next day, the lungs were briefly rinsed in PBS and the right lobe was put in 5 ml of 1% Triton X-100 in PBS for permeabilization for 48 hr or until the tissue sank. All stainings were performed in 0.2% Triton X-100 in PBS containing 5% FBS and 2% BSA and rinses were in 0.2% Triton X-100 in PBS. Staining with anti-CC10 was for 72 hr at 4 &#176;C followed by extensive washes for approximately 6 hr. Fluorescent labeling of the primary antibody was achieved with anti-goat-Alexa Fluor-594 secondary antibody overnight&#160;at 4 &#176;C followed by extensive washes for 6 hr to overnight. The following day, stained lobes were cleared through 50%, 70%, and 80% THF for 30 min each followed by three 30 min washes in 100% THF, followed by 20 min in DCM, followed by 30 min in DBE. Scale bars in a and b = 1 mm and in c = 100 &#181;m.
<img alt="Figure 9" src="/files/ftp_upload/51382/51382fig9v2highres.jpg" /> 
Figure 9. 3DISCO of pancreas. After perfusion of mice as described above in the protocol, the pancreas was dissected and cleared with the short protocol. The fluorescence is derived from ROSA26 LSL tdTomato recombination induced by tamoxifen treatment of mice carrying a 200 kb BAC transgene spanning the endogenous mouse glucagon locus with CreERT2 inserted into the ATG of glucagon. The arrowheads mark some of the fluorescent-labeled alpha cells in the islet. Scale bars in a, b and c = 1 mm and in d = 500 &#181;m.
<img alt="Table 1" src="/files/ftp_upload/51382/51382table1v3.jpg" /> 
Table 1. Tissue clearing protocols for various tissues. Examples of tissue clearing protocols for different tissues. Note that the clearing time for each step can be shortened or extended as needed to improve the clearing performance. The approximate weight of small tissues is ~20-100 mg and brain is ~300-500 mg. <a href="https://www.jove.com/files/ftp_upload/51382/51382table1.jpg" target="_blank">Please click here to view a larger version of this table.</a>

Watch this Scientific Journal Video about Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO at JoVE.com

Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO

To obtain high-resolution images of fluorescently labeled cells within large tissues, ideally, the biological samples should be imaged without sectioning. 3DISCO is a straightforward tissue clearing procedure based on sequential incubation with organic solvents. Upon clearing, the organs become transparent allowing an end-to-end laser scan of the specimen.

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact ...

imaging-cleared-intact-biological-systems-at-cellular-level

Research

JoVE Journal

Neuroscience

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.3,4,5

In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.6

In this article, we examine the effects of visually relevant state dependency on TMS induced motive phosphenic presentations.

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the organ before imaging.1,2 However, for organs that contain fluorescent proteins such as GFP and YFP, optical clearing protocols that use methanol dehydration and clear using benzyl alcohol:benzyl benzoate (BABB) while unprotected from light3 do not preserve the fluorescent signal. The protocol presented here is a novel way in which to perform whole organ optical clearing on mouse brain while preserving the fluorescence signal of YFP expressed in neurons. Altering the optical clearing protocol such that the organ is dehydrated using an ethanol graded series has been found to reduce the damage to the fluorescent proteins and preserve their fluorescent signal for multiphoton imaging.4 Using an optimized method of optical clearing with ethanol-based dehydration and clearing by BABB while shielded from light, we show high-resolution multiphoton images of yellow fluorescent protein (YFP) expression in the neurons of a mouse brain more than 2 mm beneath the tissue surface.

Multiphoton microscopy of whole mouse organs is possible by optically clearing the organ before imaging, but not all protocols preserve the fluorescent signal of fluorescent proteins. Using an optical clearing method with ethanol-based dehydration and benzyl alcohol:benzyl benzoate clearing, we show high-resolution multiphoton images of whole mouse brain expressing YFP.

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the ...

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

Research into the design and utilization of brain-implanted microdevices, such as microelectrode arrays, aims to produce clinically relevant devices that interface chronically with surrounding brain tissue. Tissue surrounding these implants is thought to react to the presence of the devices over time, which includes the formation of an insulating "glial scar" around the devices. However, histological analysis of these tissue changes is typically performed after explanting the device, in a process that can disrupt the morphology of the tissue of interest.
Here we demonstrate a protocol in which cortical-implanted devices are collected intact in surrounding rodent brain tissue. We describe how, once perfused with fixative, brains are removed and sliced in such a way as to avoid explanting devices. We outline fluorescent antibody labeling and optical clearing methods useful for producing an informative, yet thick tissue section. Finally, we demonstrate the mounting and imaging of these tissue sections in order to investigate the biological interface around brain-implanted devices.

Here we present a histological method for capturing, labeling, optically clearing, and imaging the intact brain tissue interface around chronically implanted microdevices in rodent brain tissue. Results from the techniques comprising this method are useful for understanding the impact of various penetrating brain-implants on their surrounding tissue.

Research  into the design and utilization of brain-implanted microdevices, such as  microelectrode arrays, aims to produce clinically relevant devices ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...