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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A., Holmes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+in+neurodegenerative+disease.">Microglia in neurodegenerative disease.</a> Nature reviews. Neurology. 6, 193-201 (2010).</li><li>Maragakis, N. J., Rothstein, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+Disease:+astrocytes+in+neurodegenerative+disease.+Nature+clinical+practice.">Mechanisms of Disease: astrocytes in neurodegenerative disease. Nature clinical practice.</a> Neurology. 2, 679-689 (2006).</li><li>Jahrling, N., Becker, K., Dodt, H. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-reconstruction+of+blood+vessels+by+ultramicroscopy.">3D-reconstruction of blood vessels by ultramicroscopy.</a> Organogenesis. 5, 145-148 (2009).</li><li>Spalteholz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> &#220;ber das Durchsichtigmachen von menschlichen und tierischen Pr&#228;paraten. , (1903).</li><li>Smith, C., 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=The+neuroinflammatory+response+in+humans+after+traumatic+brain+injury.">The neuroinflammatory response in humans after traumatic brain injury.</a> Neuropathology and applied neurobiology. 39, 654-666 (2012).</li><li>Zotova, E., 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=Microglial+alterations+in+human+Alzheimer's+disease+following+Abeta42+immunization.">Microglial alterations in human Alzheimer's disease following Abeta42 immunization.</a> Neuropathology and applied neurobiology. 37, 513-524 (2011).</li><li>Frewin, B., Chung, M., Donnelly, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bilateral+cochlear+implantation+in+Friedreich's+ataxia:+A+case+study.">Bilateral cochlear implantation in Friedreich's ataxia: A case study.</a> Cochlear implants international. 14, 287-290 (2013).</li><li>Kim, S. Y., Chung, K., 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=Light+microscopy+mapping+of+connections+in+the+intact+brain.">Light microscopy mapping of connections in the intact brain.</a> Trends in cognitive sciences. 17, 596-599 (2013).</li><li>Luo, X., 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+evaluation+of+retinal+ganglion+cell+axon+regeneration+and+pathfinding+in+whole+mouse+tissue+after+injury.">Three-dimensional evaluation of retinal ganglion cell axon regeneration and pathfinding in whole mouse tissue after injury.</a> Exp Neurol. 247, 653-662 (2013).</li><li>Soderblom, C., 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=Perivascular+fibroblasts+form+the+fibrotic+scar+after+contusive+spinal+cord+injury.">Perivascular fibroblasts form the fibrotic scar after contusive spinal cord injury.</a> J Neurosci. 33, 13882-13887 (2013).</li><li>McKinney, S. 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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><tbody><tr><td>Thy-1 GFP (GFP-M) mouse</td><td></td><td></td><td>Can be obtained from Jackson.</td></tr><tr><td>CX3CR1 GFP mouse</td><td></td><td></td><td>Can be obtained from Littman lab at NYU.</td></tr><tr><td>TgN(hGFAP-ECFP) mouse</td><td></td><td></td><td>Can be obtained from Jackson.</td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Mallinckrodt</td><td>7917-16</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Mallinckrodt</td><td>7892-04</td><td></td></tr><tr><td>2-2-2 Tribromoethanol</td><td>Sigma</td><td>T48402</td><td></td></tr><tr><td>2-Methyl-2-butanol</td><td>Sigma</td><td>152463</td><td>Used to prepare Avertin solution.</td></tr><tr><td>Saline</td><td>Braun</td><td></td><td></td></tr><tr><td>Tetrahydrofuran</td><td>Sigma</td><td>186562-12X100ML</td><td></td></tr><tr><td>Dichloromethane</td><td>Sigma</td><td>270997-12X100ML</td><td></td></tr><tr><td>Dibenzyl ether</td><td>Sigma</td><td>Sigma, 108014-1KG</td><td></td></tr><tr><td>Benzyl alcohol</td><td>Sigma</td><td>305197-100ML</td><td></td></tr><tr><td>Benzyl benzoate</td><td>Sigma</td><td>B6630-250ML</td><td></td></tr><tr><td>Lectin FITC</td><td>Sigma</td><td>L0401-1MG</td><td></td></tr><tr><td>anti-CC10</td><td>Santa Cruz</td><td>SC-9772</td><td>0.388888889</td></tr><tr><td>Heparin</td><td>APP pharmaceuticals</td><td>504001</td><td>Used in perfusion.</td></tr><tr><td>Glass vials</td><td>VWR</td><td>548-0554</td><td></td></tr><tr><td>Forceps</td><td>FST</td><td>11251-10</td><td></td></tr><tr><td>Mini Rotator</td><td>VWR</td><td>100498-878</td><td></td></tr><tr><td>Aluminum Foil</td><td>Fisherbrand</td><td>01-213-104</td><td></td></tr><tr><td>100 ml Media Storage Bottles</td><td>Kimax Media Bottles</td><td>EW-34523-00</td><td></td></tr><tr><td>Minipuls evolution perfusion pump with MF4* medium flow pump head</td><td>Gilson, Inc</td><td>F110701 and F117606</td><td></td></tr><tr><td>Microscopy slide</td><td>VWR</td><td>48311-703</td><td></td></tr><tr><td>FastWell</td><td>Grace Bio-Labs</td><td>FW20</td><td></td></tr><tr><td>Cover slip</td><td>Corning</td><td>2940-245</td><td></td></tr><tr><td>Glass petri dish</td><td>Corning Pyrex</td><td>3160-101</td><td></td></tr><tr><td>Dental cement</td><td>Lang Dental</td><td>1223PNK</td><td></td></tr><tr><td>Quantofix Peroxide Test Strips</td><td>Sigma</td><td>37206</td><td></td></tr><tr><td>Amira with RT resolve microscopy module</td><td>Visage Imaging</td><td>image analysis software</td><td></td></tr><tr><td>Imaris</td><td>Bitplane imaging</td><td>image analysis software</td><td></td></tr><tr><td>ImageJ</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://jove.unicartagenaproxy.elogim.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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

26.5K Views.  Genentech, Inc.. 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. 

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

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 ...

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.

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 ...

Three-Dimensional Imaging of the Vertebral Lymphatic Vasculature and Drainage using iDISCO+ and Light Sheet Fluorescence Microscopy

The lymphatic system associated with the central nervous system (CNS) includes the lymphatic vasculature that spins around the brain, the spinal cord, and its associated LNs. The CNS-associated lymphatic system is involved in the drainage of CSF macromolecules and meningeal immune cells toward CNS-draining LNs, thereby regulating waste clearance and immune surveillance within CNS tissues. Presented is a novel approach to obtain three-dimensional (3D) and cellular resolution images of CNS-associated lymphatics while preserving the integrity of their circuits within surrounding tissues. The iDISCO+ protocol is used to immunolabel lymphatic vessels in decalcified and cleared whole mount preparations of the vertebral column that are subsequently imaged with light sheet fluorescence microscopy (LSFM). The technique reveals the 3D structure of the lymphatic network connecting the meningeal and epidural spaces around the spinal cord to extravertebral lymphatic vessels. Provided are 3D images of the drainage circuits of molecular tracers previously injected into either the CSF via the cisterna magna or the thoracolumbar spinal parenchyma. The iDISCO+/LSFM approach brings unprecedented opportunities to explore the structure and function of the CNS-associated lymphatic system in neurovascular biology, neuroimmunology, brain and vertebral cancer, or vertebral bone and joint biology.

Three-Dimensional Imaging of the Vertebral Lymphatic Vasculature and Drainage using iDISCO+ and Light Sheet Fluorescence Microscopy

A protocol is presented combining tissue clearing with light sheet fluorescence microscopy (LSFM) to obtain three-dimensional and cellular resolution images of the lymphatic vessels and lymph nodes (LNs) collecting the cerebrospinal fluid (CSF) and spinal epidural fluid.

The lymphatic system associated with the central nervous system (CNS) includes the lymphatic vasculature that spins around the brain, the spinal cord, and ...

Immunology and Infection

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 ...

Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of structural changes caused by genetic or environmental perturbations. Whole-brain imaging results in more accurate quantification of cells and the study of region-specific differences that may be missed with commonly used microscopy of physically sectioned tissue. Using light-sheet microscopy to image cleared brains greatly increases acquisition speed as compared to confocal microscopy. Although these images produce very large amounts of brain structural data, most computational tools that perform feature quantification in images of cleared tissue are limited to counting sparse cell populations, rather than all nuclei.
Here, we demonstrate NuMorph (Nuclear-Based Morphometry), a group of analysis tools, to quantify all nuclei and nuclear markers within annotated regions of a postnatal day 4 (P4) mouse brain after clearing and imaging on a light-sheet microscope. We describe magnetic resonance imaging (MRI) to measure brain volume prior to shrinkage caused by tissue clearing dehydration steps, tissue clearing using the iDISCO+ method, including immunolabeling, followed by light-sheet microscopy using a commercially available platform to image mouse brains at cellular resolution. We then demonstrate this image analysis pipeline using NuMorph, which is used to correct intensity differences, stitch image tiles, align multiple channels, count nuclei, and annotate brain regions through registration to publicly available atlases.
We designed this approach using publicly available protocols and software, allowing any researcher with the necessary microscope and computational resources to perform these techniques. These tissue clearing, imaging, and computational tools allow measurement and quantification of the three-dimensional (3D) organization of cell-types in the cortex and should be widely applicable to any wild-type/knockout mouse study design.

This protocol describes methods for conducting magnetic resonance imaging, clearing, and immunolabeling of intact mouse brains using iDISCO+, followed by a detailed description of imaging using light-sheet microscopy, and downstream analyses using NuMorph.

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of ...

ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in biology. Transitional methods require a substantial amount of time and effort to obtain a 3D image and including sectioning the intact tissue, immunolabeling, and imaging serially-sectioned tissue, which produces a loss of information at each step of the process. In recently developed approaches for high-resolution imaging within intact tissue, molecular characterization has been restricted to the labeling of proteins. However, currently available protocols for organ clearing require a considerably long process time, making it difficult to implement tissue clearing techniques in the lab. We recently established a rapid and highly-reproducible protocol termed ACT-PRESTO (active clarity technique&#8211;pressure related efficient and stable transfer of macromolecules into organs), which allows for tissue clearance within several hours. Moreover, ACT-PRESTO enables rapid immunolabeling with conventional methods and accelerates antibody penetration into the deep layer of densely-formed, thick specimens by applying pressure or convection flow. We describe how to prepare tissues, how to clear by lipid removal using electrophoresis, and how to immuno-stain by a pressure-assisted delivery. The rapidity and consistency of the protocol will expedite the performance of 3D histological research and volume-based diagnoses.

ACT-PRESTO (active clarity technique&#8211;pressure related efficient and stable transfer of macromolecules into organs) enables rapid, efficient, but inexpensive tissue clearing and rapid antibody penetration into cleared tissues by passive diffusion or pressure-assisted delivery. Using this method, a wide range of tissues can be cleared, immunolabeled by multiple antibodies, and volume-imaged.

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in ...

Biology

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

Imaging the Gut with "CLARITY"

CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) has recently evolved as a valuable technique involving acrylamide embedding to delipidate tissue (without sectioning) and to preserve the 3-D tissue structure for immunostaining. The technique is highly relevant in imaging the dynamic gut environment where different cell types interact during homeostasis and disease states. This method optimized for the mouse gut is described here, which helps to trace cell types like epithelia, enteroendocrine, neurons, glia, and the neuronal projections into the epithelia or enteroendocrine cells that mediate microbial sensing or nutrient chemo sensing respectively. The gut tissue (1-1.5 cm) is fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at 4 &#176;C overnight on day 1. On day 2, PFA is discarded, and the tissue is washed thrice with PBS. The tissue is hydrogel embedded to preserve its integrity by incubation in 4% hydrogel (acrylamide) solution in PBS (diluted from 30% ProtoGel) overnight at 4 &#176;C. On day 3, the tissue-hydrogel solution is incubated at 37&#160;&#176;C for 1 h to allow hydrogel polymerization. Tissue is then washed thrice gently with PBS to remove excess hydrogel. The subsequent step of delipidation (clearing) involves tissue incubation in sodium dodecyl sulfate (8% SDS in PBS) at 37 &#176;C for 2 days (days 4 &#38; 5) on a shaker at room temperature (RT). On day 6, the cleared tissue is thoroughly washed with PBS to remove SDS. Tissue can be immunostained by incubation in primary antibodies (diluted in 0.5% normal donkey serum in PBS containing 0.3% Triton X-100), overnight at 4&#176;C, and subsequent incubation in appropriate secondary Alexa Fluor antibodies for 1.5 h at RT, and nuclear staining with DAPI (1: 10000). The tissue is transferred onto a clean glass slide and mounted using VectaShield for confocal imaging.

We describe the protocol for passive CLARITY (PACT) staining of mouse intestine to enable visualization of subepithelial tissues, including neurons, glia and enteroendocrine cells (EEC) without tissue sectioning. The protocol involves hydrogel embedding of formaldehyde-fixed tissue, and subsequent delipidation using an anionic detergent to "clear" the tissue.

CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) has recently evolved as a valuable technique involving ...

3D Visualization of Immune Cell Populations in HIV-Infected Tissues via Clearing, Immunostaining, Confocal, and Light Sheet Fluorescence Microscopy

Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Syndrome (AIDS), is a major global health concern with nearly 40 million individuals infected worldwide and no widely accessible cure. Despite intensive efforts, a detailed understanding of virus and host cell interactions in tissues during infection and in response to therapy remains incomplete. To address these limitations, water-based tissue clearing techniques CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) and CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/in situ-hybridization-compatible Tissue hYdrogel) are applied to visualize complex virus host-cell interactions in HIV-infected tissues from animal models and humans using confocal and light sheet fluorescence microscopy. Optical sectioning of intact tissues and image analysis allows rapid reconstruction of spatial information contained within whole tissues and quantification of immune cell populations during infection. These methods are applicable to most tissue sources and diverse biological questions, including infectious disease and cancer.

Tissue clearing, combined with immunofluorescence microscopy, allows spatial visualization and quantification of immune-cell populations and virus proteins within intact tissues. Optical sectioning of cleared tissues with confocal and light sheet fluorescence microscopy can generate 3D models of complex tissue environments and reveal spatial heterogeneity exhibited during HIV infection.

Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Syndrome (AIDS), is a major global health concern with nearly 40 ...

Optimized Tissue-Clearing Protocols for 3D Imaging of the Murine Aorta 

Three-Dimensional Imaging of Aortic Tissues in Atherosclerosis

Recent research has advanced the understanding of atherosclerosis as a transmural chronic inflammatory disease involving all three layers of the arterial wall, including the intima plaque, the media, and the adventitia, which forms the outer connective tissue coat of arteries. Our recent studies have suggested that the adventitia is used by the peripheral nervous system as a conduit for reaching all tissue cells. We also found that the peripheral nervous system, that is, the sensory and sympathetic nervous system, undergoes major remodeling processes involving the neogenesis of axon networks adjacent to atherosclerotic plaques. In this context, understanding the structure of the neural network and its interactions with vascular components of diseased arteries holds major promises for a better understanding of cardiovascular disease pathogenesis. To achieve these objectives, methods to visualize the subcellular architecture of the intact healthy and diseased arteries together with their surrounding perivascular compartments are needed. Tissue clearing allows intact deep-tissue imaging of larger tissue compartments that are otherwise inaccessible. It allows volumetric imaging of intact arteries through the integration of labeling, clearing, advanced microscopic imaging, and image processing tools. Here, we describe two distinct but complementary passive tissue clearing approaches, that is, aqueous-based 2, 2-thiodiethanol (TDE) clearing and solvent-based immunolabeling-enabled three-dimensional imaging of solvent-cleared organ (iDISCO) clearing to image isolated aortic segments or whole aorta in-situ in the whole mouse.

Here, we present optimized tissue-clearing protocols to image the murine aorta in three dimensions (3D). We delineate state-of-the-art procedures for immunostaining, optical clearing, and imaging with the intent to define the anatomical proximity of the peripheral nervous system with atherosclerotic plaques and the adventitia in atherosclerosis.

Recent research has advanced the understanding of atherosclerosis as a transmural chronic inflammatory disease involving all three layers of the arterial ...