Name: novel passive clearing methods for the rapid production of optical transparency in whole cns tissue
Uploaded: 2018-05-08T14:00:00.000+00:00
Duration: 6 min 14 s
Description: Watch this Scientific Journal Video about Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue at JoVE.com

This work was supported by the Brain Korea 21 PLUS Project for Medical Science, Yonsei University. In addition, this work was supported by a grant from the National Research Foundation of Korea (NRF-2017R1D1A1B03030315).

All procedures have been approved by the appropriate research ethics committee at Yonsei University College of Medicine. All experimental animals are sacrificed in accordance with the guidelines of the laboratory animal care committee at Yonsei University College of Medicine.
1. Preparation of Reagents
Caution: Paraformaldehyde (PFA), acrylamide and sodium dodecyl sulfate (SDS) are toxic irritants and thus should be handled in a fume hood with appropriate personal protective equipment (PPE; lab coat, gloves, protective eyewear).
<ol>
	<li>4% acrylamide (A.A) solution (A4P0): Add 20 mL of 40% acrylamide solution to 180 mL of 0.1 M phosphate-buffered saline (PBS).</li>
	<li>0.25% VA-044 solution: Add 0.5 g of 2,2&#180;-Azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044) powder to 200 mL of 0.1 M phosphate-buffered saline (PBS). While the VA-044 powder can be stored at room temperature, it must be stored at 4 &#176;C upon solubilization in PBS. For best results, prepare only the amount of solution needed for the experiment.</li>
	<li>PACT cocktail solution: Add 10 g of acrylamide to 225 mL of 4% paraformaldehyde (PFA) solution and adjust the volume to 200 mL with 4% PFA. Prior to use, add 100 mg of VA-044 powder to 40 mL of mixture in a 50 mL conical tube.</li>
	<li>PACT clearing solution buffer: Add 40 g of sodium dodecyl sulfate (SDS) to 350 mL of 0.1 M PBS and adjust the volume to 500 mL with 0.1 M PBS. For mPACT clearing solution, add 0.5% &#945;-thioglycerol to the PACT clearing solution buffer.</li>
	<li>The mice used in this study were 2-week-old BALB male mice; the rats used in this study were 2 week old male SD-rats.</li>
</ol>
2. Anesthesia and Perfusion Surgery
Caution: PFA and acrylamide are toxic irritants and thus should be handled in a fume hood with appropriate PPE.
<ol>
	<li>Anesthetize the animal in a pathogen-free room with 30 mg/kg of zoletil using a 1 mL syringe with a 26-gauge needle. Monitor the animal for 5 -10 min.</li>
	<li>Once the animal has reached a surgical plane of anesthesia, use the toe-pinch response method to determine the depth of anesthesia. Confirm unresponsiveness. 
	NOTE: The following steps involving mouse and rat surgery follow a similar protocol used in a previous study11.</li>
	<li>Make a 5-6 cm incision beneath the rib cage, through the integument and abdominal wall.</li>
	<li>Use curved, blunt scissors to make an incision in the diaphragm, palpating the thoracic area to locate the site of incision beforehand.</li>
	<li>Extend the incision across the entire length of the ribcage to expose the pleural cavity.</li>
	<li>Carefully displace the lungs. Make a cut through the ribcage up to the collarbone. Using iris scissors, make a small incision to the posterior end of the left ventricle. Insert an 18-gauge blunt- or olive-tipped perfusion needle into the ascending aorta, passing through the cut ventricle.</li>
	<li>Secure the needle and prevent leakage by clamping the heart with a hemostat. Alternatively, use a modified hemostat to clamp the aorta around the needle tip.</li>
	<li>Make a large incision in the right atrium, taking care to ensure minimal damage to the descending aorta. The rat is now ready for perfusion.</li>
</ol>
3. Whole Perfusion and Dissection of Rat
Caution: PFA and acrylamide are toxic irritants and thus should be handled in a fume hood with appropriate PPE.
NOTE: The following whole perfusion steps are a similar to a protocol used in a previous study by Woo et al. (2016)10,11.
<ol>
	<li>Attach the right atrium of heart to an 18-gauge needle, taking care to not introduce any air bubbles.</li>
	<li>Using a 50 mL syringe, quickly and evenly pump 50 mL of cold 0.1 M PBS solution containing heparin (10 units/mL).</li>
	<li>Connect the 18-gauge needle to the tube of the peristaltic pump.</li>
	<li>Wash with 200 mL of cold 0.1 M PBS solution containing heparin (10 units/mL) at a circulation velocity of 10 mL/min.</li>
	<li>Fix with 250 mL of cold 4% PFA solution at a circulation velocity of 10 mL/min. The rat should be stiff at this stage.</li>
	<li>Collect and store the remaining PFA solution for disposal.</li>
	<li>For PACT, perfuse tissues with a chilled PACT cocktail solution of 4% PFA, 4% acrylamide, and 0.25% VA-044 powder with an 18-gauge needle, and then remove the head and spine. Expose the skull by making a midline incision from the neck to the nose. This additional perfusion step is unnecessary for the psPACT and mPACT methods; after fixation, immediately isolate the head and spine and expose the skull.</li>
	<li>Expose the base of the skull by removing any residual neck and spine muscle.</li>
	<li>Use rongeurs and scissors to peel away the skull. Remove the brain and spinal cord.</li>
	<li>Store the tissue in 4% PFA at 4 &#176;C; tissues can be stored for up to 1 week.</li>
</ol>
4. Hydrogel Monomer Infusion and Polymerization of the Rat and Mouse CNS
Caution: Acrylamide, SDS, &#945;-thioglycerol and PFA are irritants and thus should be handled in a fume hood and with appropriate PEE.
<ol>
	<li>PACT (Passive Clearing Technique)
	<ol>
		<li>Isolate and culture the whole CNS (brain and spinal cord) from fixed mice and rats to a 50 mL tube containing a chilled PACT cocktail solution of 4% PFA, 4% acrylamide, and 0.25% VA-044 powder and store at 4 &#176;C for 24 h. Ensure that the tissue is completely immersed in the solution.</li>
		<li>Embed the sample in nitrogen gas for 10 min using a tissue gel hybridization system connected to a nitrogen tank: Set the system to 37 &#176;C. Transfer tissues to a 50 mL tube containing fresh cold (4 &#176;C) PACT cocktail solution and connect with tube cap, then turn on the vacuum.</li>
		<li>To polymerize the hydrogel, place the tube containing the sample in a shaking incubator (150 rpm, 37 &#176;C) for 3 h, or until polymerization is complete.</li>
		<li>Using blotting paper (see Table of Materials), remove the remaining polymerized hydrogel surrounding the tissues.</li>
		<li>Transfer the tissue to a 50 mL tube containing clearing solution (8% SDS in 0.1 M PBS, pH 8.0).</li>
		<li>Place the sample in a shaking incubator set to 37 &#176;C and 150 rpm until the tissue has been cleared. On average, it takes about 20 days for the mouse CNS to achieve full clarity.</li>
	</ol>
	</li>
	<li>psPACT (process separate Passive Clearing Technique)
	<ol>
		<li>Isolate the whole CNS (brain and spinal cord) with bone cutter and scissors from PFA-fixed mice and rats on a clean bench.</li>
		<li>Transfer tissues to a 50 mL tube containing 4% PFA, and store at 4 &#176;C for 24 h. Ensure that the tissue is completely immersed in the fixative.</li>
		<li>Wash the fixed tissue for 1 h in 0.1 M PBS, and then transfer to A4P0 solution (4% acrylamide in 0.1 M PBS). Store at 37 &#176;C for 24 h.</li>
		<li>Wash the tissue for 5 min in 0.1 M PBS.</li>
		<li>Immerse the tissue in 0.25% VA-044 in 0.1 M PBS. Store at 37 &#176;C for 6-24 h, and then transfer to fresh 0.25% VA-044/PBS solution.</li>
		<li>Embed the sample in nitrogen gas for 10 min using a tissue gel hybridization system (see Table of Materials) connected to a nitrogen tank: Transfer tissues to a 50 mL tube containing fresh cold (4 &#176;C) 0.25% VA-044/PBS solution, and connect with tube cap. Turn on the vacuum.</li>
		<li>Transfer the tissue to clearing solution (8% SDS in 0.1 M PBS, pH 8.0).</li>
		<li>Incubate the sample in a shaking incubator set to 37 &#176;C and 150 rpm until the tissue has been cleared. On average, it takes approximately 17 days for mouse CNS to achieve full clarity.</li>
	</ol>
	</li>
	<li>mPACT (modified Passive Clearing Technique)
	<ol>
		<li>Isolate the whole CNS (brain and spinal cord) with bone cutter and scissors from PFA-fixed mice and rats on a clean bench.</li>
		<li>Follow steps 4.2.2 - 4.2.8 of the psPACT protocol.</li>
		<li>Transfer the tissue to clearing solution. Note that unlike the PACT and psPACT protocols, mPACT requires a clearing solution consisting of 0.5% &#945;-thioglycerol in addition to 8% SDS in 0.1 M PBS, pH 8.0. &#945;-thioglycerol is an un-browning agent that helps clear tissue more rapidly and effectively. Ensure that the tissue is fully immersed in the solution.</li>
		<li>Place the sample in a shaking incubator set to 37 &#176;C and 150 rpm until the tissue has been cleared. On average, it takes approximately 2 weeks for mouse CNS to achieve full clarity.</li>
	</ol>
	</li>
</ol>
5. Refractive Index Matching and Immunostaining of Cleared CNS
NOTE: nRIMS (Nycodenz-based Refractive Index Matching Solution) consists of 0.8 g/mL Nycodenz powder dissolved in 30 mL base buffer (0.01% sodium azide and Tween-20 in 0.1 M PBS, pH 8.0). It is recommended that the solution is placed in a 37 &#176;C shaking incubator to allow for proper solvation of the powder.
<ol>
	<li>Incubate tissues in 0.1% Triton X-100 in 0.1 M PBS for 2 h, then block with 2% bovine serum albumin (BSA) in 0.1 M PBS for 6 h.</li>
	<li>Wash sections three times in PBST (0.1% Tween-20 in 0.1 M PBS) for 2 h. Stain sections with primary antibodies (in this case, an anti-rabbit PECAM-CD31 antibody, which stains blood vessels) for 2 days.</li>
	<li>Stain sections with secondary antibodies (in this case, a goat anti-rabbit IgG Cy3 fluorescent conjugate) in 2% BSA for 2 days.</li>
	<li>Wash labeled tissues three times in PBST for 2 h, and store in 15 mL nRIMS for 2 - 10 days.</li>
	<li>Before imaging, move labeled tissues to a small amount of nRIMS on 35- or 60-mm tissue culture dishes. Fix with silicone around the bottom edge of the dish.</li>
	<li>Add 1.5 - 2 mL of fresh nRIMS.</li>
</ol>
6. Image processing
<ol>
	<li>Acquire images of cleared tissues with tile scanning using a confocal laser-scanning microscopewith 10x magnification (see Table of Materials). For of Cy3-labeled tissue, use wavelengths of 550-600 nm.</li>
</ol>

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The authors have nothing to disclose.

While the passive, non-electrophoretic extraction methods employed in PACT significantly improved the consistency achieved with previous tissue clearing methods such as CLARITY2,3,4,7,8, the technique still bears several shortcomings, the most pressing of which is the length of time required to achieve maximal tissue clarity12. In the current study, we present modified PACT protocols that significantly reduce the time required for tissue clearing while still preserving tissue integrity and structure6,10. Furthermore, for the first time, we demonstrate the application of tissue clearing protocols in whole CNS tissues and in a higher-order rodent model, the rat10.
Specifically, psPACT involves sample processing in 4% acrylamide and 0.25% VA-044 in two separate steps during hydrogel formation, while mPACT further builds upon psPACT and supplements the 8% SDS clearing solution with 0.5% &#945;-thioglycerol10. Separating the acrylamide and subsequent VA-044 step avoids the need for removing remaining unpolymerized hydrogel monomers that surround the tissue, which can compromise final tissue integrity after achieving transparency with PACT; additionally, this significantly reduces the time needed to produce maximal optical clearance. The addition of &#945;-thioglycerol in the mPACT method further accelerates the clearing process13, as &#945;-thioglycerol is an un-browning agent that helps clear regions of the tissue that are less accessible by using only the reagents in the original PACT method7,8,9.
A comparison of PACT, psPACT and mPACT on rodent tissue demonstrated that while psPACT improves optical transparency relative to the original PACT protocol, mPACT most efficiently allows for the clearing and subsequent high-resolution imaging of both whole mammalian organs and sectioned slices, as demonstrated in Figure 2, Figure 3, Figure 4. The &#945;-thioglycerol-based lipid washout of processed organs in the mPACT method proved key to the differences in transparency, efficiency, and tissue stability in comparison to the other two clearing methods. This allowed for the rapid 3D analysis of processed organs, specifically with respect to blood vasculature morphology, via immunofluorescence and confocal microscopy (Figure 5D,E).
While the addition of &#945;-thioglycerol to the SDS-based clearing solution is critical for accelerating optical transparency, it also slightly increases tissue fragility and makes it more susceptible to damage post-processing. The clear superiority of mPACT with regards to its efficiency outweighs this limitation; nevertheless, work is currently being done to ensure that maximal clearing can be achieved with minimal to no expense of tissue integrity.
Thus, our results establish mPACT as a powerful investigative tool for providing a 3D analysis of the functional and anatomical features of mammalian organs, including but not limited to neuronal networks, blood vasculature, and tissue matrix architecture. Moving forward, we believe mPACT could be especially useful in the investigation of higher-order biological mechanisms that contribute to the pathophysiology of disease, such as tissue injury, developmental malformations, cancer, and neurodegenerative disease14,15,16,17. Combined with a study of the finer cellular mechanisms involved in disease, mPACT has the potential to provide a more complete, multi-scale understanding of how various disparate disease mechanisms interact to give rise to the observed phenotype.

A fundamental objective of scientific and clinical inquiry involves attaining a complete understanding of organ structure and function; however, the exceedingly complex nature of mammalian organs often serves as a barrier to fully achieving this aim1. CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tisssue-hYdrogel)2,3,4, which involves building an acrylamide-based hydrogel hybrid from intact tissues, achieves optical clearance of a variety of organs, including the brain, liver, and spleen, while preserving their structural integrity5. CLARITY has thus enabled not only visualization but also the opportunity to finely dissect complex cellular networks and tissue morphologies without the need for sectioning.
In order to achieve tissue clearance, CLARITY employs electrophoretic methods to remove the lipid content of the sample at hand. While CLARITY has been noted for producing physically stable tissue-hydrogel hybrids, studies have shown that its use of electrophoretic tissue clearing (ETC) methods yields variable results in terms of tissue quality, including browning, epitope damage, and protein loss5,6. To address these issues, modified protocols such as PACT (PAssive Clarity Technique), which replaces the ETC treatment with a passive, ionic-detergent based delipidation technique, have been developed7,8,9. Despite achieving a greater consistency in results, however, PACT requires more time to obtain maximal clearance. Furthermore, none of these techniques have yet been applied to the whole CNS form, or in larger rodent models such as rats and guinea pigs.
The present study seeks to address these limitations by proposing novel methodologies, psPACT (process-separate PACT) and mPACT (modified PACT), for facilitating the fast clearance of the whole CNS and internal organs in both mouse and rat models10. Specifically, psPACT processes tissues in 4% acrylamide and 0.25% VA-044 in two separate steps during hydrogel formation; mPACT essentially involves the same steps, but supplements the SDS-based clearing solution with 0.5% &#945;-thioglycerol as a key reagent. Both techniques harness the endogenous systemic and cerebrospinal circulatory systems to significantly reduce the time needed to produce optical clearance. As a proof of principle, we demonstrate the use of confocal microscopy to analyze blood vessel patterns in the cleared tissues10.

<table><tbody><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Affymetrix, Inc.</td><td>75819</td><td>Clearing solution</td></tr><tr><td>Nycodenz</td><td>Axia-Shield</td><td>1002424</td><td>nRIMS solution</td></tr><tr><td>40% Acrylamide Solution</td><td>Bio Rad Laboratories, Inc.</td><td>161-0140</td><td>Polymerization (A4P0)</td></tr><tr><td>2,2&amp;acute;-Azobis[2-(2-imidazolin-2-yl)propane] Dihydrochloride</td><td>Wako Pure Chemical Industries, Ltd.</td><td>017-19362</td><td>Polymerization (VA-044)</td></tr><tr><td>1-Thioglycerol</td><td>Sigma-Aldrich</td><td>M1753-100ML</td><td>Clearing solution (mPACT)</td></tr><tr><td>Tween-20</td><td>Georgiachem</td><td>9005-64-5</td><td>nRIMS solution</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787-50ML</td><td>Immuno Staining</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Bovogen</td><td>BSA100</td><td>Immuno Staining</td></tr><tr><td>Heparin</td><td>Merck Millipore</td><td>375095</td><td>Perfusion (PBS)</td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S2002-25G</td><td>nRIMS solution</td></tr><tr><td>PECAM-CD31 antibody</td><td>Santa Cruz Biotechnology Inc.</td><td>sc-28188</td><td>Immuno Staining</td></tr><tr><td>Goat anti-rabbit-IgG Cy3 fluorescent conjugate</td><td>Jackson ImmunoResearch Inc.</td><td>111-165-003</td><td>Immuno Staining</td></tr><tr><td>4% Paraformaldehyde</td><td>Tech &amp;amp; Innovation</td><td>BPP-9004</td><td>Perfusion, Polymerization</td></tr><tr><td>20X Phosphate Buffered Saline (pH 7.4)</td><td>Tech &amp;amp; Innovation</td><td>BPB-9121</td><td>Perfusion, Buffer</td></tr><tr><td>10 mL stripette</td><td>Coatar</td><td>4488</td><td>Solution transfer</td></tr><tr><td>50 mL tube</td><td>Falcon</td><td>352070</td><td>Clearing tube</td></tr><tr><td>35 mm Cell culture dish</td><td>SPL</td><td>20035</td><td>Imaging</td></tr><tr><td>Confocal dish</td><td>SPL</td><td>211350</td><td>Imaging</td></tr><tr><td>1 mL syringe</td><td>Korea vaccine Co., Ltd</td><td>26G 1/2</td><td>Anesthetize&amp;nbsp;</td></tr><tr><td>50 mL syringe</td><td>Korea vaccine Co., Ltd</td><td>21G1 1/4</td><td>Perfusion</td></tr><tr><td>Acrylamide</td><td>Sigma-Aldrich</td><td>A3553</td><td>Polymerization (A4P0)</td></tr><tr><td>Whatman 3MM paper</td><td>Sigma-Aldrich</td><td>Z270849</td><td>Blotting paper for gel removal</td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td>LSM780</td><td>Imaging</td></tr><tr><td>ZEN lite Software</td><td>Zeiss</td><td>ZEN 2012</td><td>Imaging</td></tr><tr><td>Peristaltic pump</td><td>Longerpump</td><td>BT100-1F</td><td>Perfusion</td></tr><tr><td>EasyGel</td><td>Lifecanvas Technologies</td><td>EasyGel</td><td>Tissue gel hybridization system</td></tr></tbody></table>

novel passive clearing methods for the rapid production of optical transparency in whole cns tissue

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, a multitude of novel clearing methodologies including CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), SWITCH (system-wide control of interaction time and kinetics of chemicals), MAP (magnified analysis of the proteome), and PACT (passive clarity technique), have been established to further expand the existing toolkit for the microscopic analysis of biological tissues. The present study aims to improve upon and optimize the original PACT procedure for an array of intact rodent tissues, including the whole central nervous system (CNS), kidneys, spleen, and whole mouse embryos. Termed psPACT (process-separate PACT) and mPACT (modified PACT), these novel techniques provide highly efficacious means of mapping cell circuitry and visualizing subcellular structures in intact normal and pathological tissues. In the following protocol, we provide a detailed, step-by-step outline on how to achieve maximal tissue clearance with minimal invasion of their structural integrity via psPACT and mPACT.

Generation of a transparent model of the whole CNS using optimized passive clearing techniques
Optical clearance of mouse and rat whole CNS tissues was rapidly achieved using various passive clearing techniques (Figure 1). A schematic of tissue clearing over time is shown in Figure 2A. Unlike the original PACT method, psPACT (process-separate PACT) involves treating samples with 4% acrylamide (A4P0) and 0.25% VA-044 initiator solution in two separate steps in order to form the hydrogel hybrid. mPACT (modified PACT) further improves upon psPACT by supplementing the 8% SDS clearing solution with 0.5% &#945;-thioglycerol. Samples processed via PACT, psPACT, and mPACT at Day 14 are compared and presented in Figure 2B.
In a previous study, we reported that while the original PACT protocol achieved optical clearance in 23 days, psPACT and mPACT cleared the entire mouse CNS in just 20 and 14 days, respectively10. In the present study, when we treated mouse brain samples via the mPACT protocol, we found that they achieved optical transparency after 14 days (Figure 2B). Rat whole CNS samples processed via psPACT and mPACT also showed that mPACT achieved maximal clearance with the greatest efficiency, as tissues were cleared in 30 and 20 days, respectively (Figure 3A, B). Adult rat brains alone, as opposed to the entire CNS, were cleared with mPACT in a mere 5 days (Figure 4A). Blood vessel patterns of cleared rat brains were analyzed via immunofluorescence after psPACT and mPACT processing to demonstrate the utility of these clearing procedures for the anatomical and functional analysis of the brain (Figure 4B, mPACT data shown in Woo et al. 2016)10.
Using these optimized passive tissue clearing protocols, we were able to visualize intact, transparent models of the whole CNS, although the clearing times differed among the three protocols. Optimized passive clearing methods achieved organ clarity in a shorter length of time. Taken together, these results suggest that the mPACT clearing method can generate clear CNS more stably and rapidly than the previous passive clearing methods. Thus, mPACT holds great potential for use in future structural and anatomical analyses of mammalian organs and provides a significant advantage over existing methods in terms of both efficacy and safety.
Generation of optically cleared internal organs and embryos using mPACT
In addition to whole CNS tissues, the adult rat spleen and kidney were cleared with mPACT in 19 and 23 days, respectively (Figure 5A,B). Mouse embryos at E10.5 and E13.5 were also cleared after 3 days of processing via the mPACT protocol. Figure 5C-E shows the visualization of blood vessel patterns in the whole embryo, as well as the microvasculature of the tail region of the mouse embryo, rat cauda equine and the rat spleen. These results suggest that mPACT can be used to generate transparent models of a wide variety of tissues and organs for 3D anatomical analysis.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57123/57123fig1.jpg" /> 
Figure 1: Schematic representation of optimized passive clearing techniques (PACT). An outline of the individual steps involved in each of the three PACT protocols is provided alongside the reagents required for each step. The original PACT protocol is tissue polymerization from hydrogel formation with a cold mixture of 4% PFA, 4% Acrylamide (A.A) solution and 0.25% VA-044 initiator solution. After tissue is polymerized using nitrogen gas, it is passively cleared with 8% SDS clearing solution. The psPACT (process-separate PACT) uses separate treatment processes of 4% A.A solution and 0.25% VA-044 initiator solution for the hydrogel formation of the tissue; these conditions differ from those of the original PACT protocol. The mPACT (modified PACT) additional adds 0.5% &#945;-thioglycerol to the 8% SDS clearing solution of the psPACT protocol. For all three protocols, the intact tissues are incubated at &#62;= 37 &#176;C in a shaking incubator until maximal clearance iss achieved. <a href="/files/ftp_upload/57123/57123fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57123/57123fig2.jpg" /> 
Figure 2: Optical transparency of mouse whole central nervous system (CNS) by passive tissue clearing. (A) Schematization of whole CNS tissue clearing via PACT-based methods over time. (B) Comparison of the three passive clearing protocols (original PACT, psPACT, mPACT) in mouse whole CNS at 14 days after processing. (C) Optical clearance of the mouse brain subjected to mPACT processing over 14 days. This figure has been modified from Woo et al. 201610. The letters in 2C are 3 mm tall. <a href="/files/ftp_upload/57123/57123fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57123/57123fig3.jpg" /> 
Figure 3: Optical transparency of rat whole CNS tissues post-psPACT and mPACT. (A) Adult rat whole CNS tissue achieved maximal optical clearance with psPACT after 30 days. (B) Adult rat whole CNS tissue achieved maximal optical clearance with mPACT after 20 days. The before and after diagrams indicate the size of the whole CNS before and after treatment, as measured by eye. <a href="/files/ftp_upload/57123/57123fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57123/57123fig4.jpg" /> 
Figure 4: mPACT processing and microvasculature visualization of sectioned adult rat brain. (A) Comparison of 4-mm thick adult rat brain sections after mPACT processing at 3 and 5 days. (B) 3D projection of blood vessel patterns in the adult rat brain visualized with anti-CD31 antibody after psPACT processing. Scale bars, 1,500 &#181;m and 100 &#181;m. This figure has been modified from Woo et al. 201610. <a href="/files/ftp_upload/57123/57123fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57123/57123fig5.jpg" /> 
Figure 5: mPACT processing of rodent organs and whole mouse embryos. mPACT achieved optical transparency in the adult rat (A) spleen and (B) kidney in 19 and 23 days, respectively. (C) mPACT produced transparent E10.5 and E13.5 mouse embryos after 3 days. (D) Visualization of microvasculature with anti-CD31 in the tail region of the mouse embryo after clearing with mPACT. (E) 3D projection of vasculature in the rat cauda equine and (F) rat spleen with anti-CD31, after processing via mPACT. This figure has been modified from Woo et al. 201610. <a href="/files/ftp_upload/57123/57123fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue at JoVE.com

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

Here, we present two novel methodologies, psPACT and mPACT, for achieving maximal optical transparency and subsequent microscopic analysis of tissue vasculature in the intact rodent whole CNS.

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

novel-passive-clearing-methods-for-rapid-production-optical

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.7K Views.  Yonsei University College of Medicine. Here, we present two novel methodologies, psPACT and mPACT, for achieving maximal optical transparency and subsequent microscopic analysis of tissue vasculature in the intact rodent whole CNS.

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

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

Rapid Optical Clearing for Semi-High-Throughput Analysis of Tumor Spheroids

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the spheroid at the cellular level is important for analysis, yet existing techniques are often expensive, laborious, use non-standard equipment, cause significant size distortion, or are limited to relatively small spheroids. This protocol presents a new method of mounting and clearing spheroids that address these issues while allowing for confocal analysis of the inner structure of spheroids. In contrast to existing approaches, this protocol provides for rapid mounting and clearing of a large number of spheroids using standard equipment and laboratory supplies. Mounting spheroids in a pH-neutral agarose-PBS gel solution before introducing a refractive-index-matched clearing solution minimizes size distortion common to other similar techniques. This allows for detailed quantitative and statistical analysis where the accuracy of size measurements is paramount. Furthermore, compared to liquid clearing solutions, the agarose gel technique keeps spheroids fixed in place, allowing for the collection of three-dimensional (3D) confocal images. The present article elaborates how the method yields high-quality two- and 3D images that provide information about inter-cell variability and inner spheroid structure.

Tumor spheroids are becoming increasingly utilized to assess tumor cell-microenvironment interactions and therapy response. The present protocol describes a robust but simple method for semi-high-throughput imaging of 3D tumor spheroids using rapid optical clearing.

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the ...

Cancer Research

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 ...

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole retina imaging techniques compared to conventional retinal flat mount preparations and sections. However, immunofluorescent imaging of intact whole retina is hindered by the opaque coatings of the eyeball, i.e., sclera, choroid, and retinal pigment epithelium (RPE) and the light scattering properties of retinal layers that prevent full thickness high resolution optical imaging. Chemical bleaching of the pigmented layers and tissue clearing protocols have been described to address these obstacles; however, currently described methods are not suitable for imaging endogenous fluorescent molecules such as green fluorescent protein (GFP) in intact whole retina. Other approaches bypassed this limitation by surgical removal of pigmented layers and the anterior segment of the eyeball allowing intact eye imaging, though the peripheral retina and hyaloid structures were disrupted. Presented here is an intact whole retina and vitreous immunofluorescent imaging protocol that combines surgical dissection of the sclera/choroid/retina pigment epithelium (RPE) layers with a modified tissue clearing method and light sheet fluorescent microscopy (LSFM). The new approach offers an unprecedented view of&#160;unperturbed vascular and neuronal elements of the retina as well as the vitreous and hyaloid vascular system in pathologic conditions.

Presented here is a protocol for intact whole retina imaging in which the outer opaque/pigmented layers of the eyeball are surgically removed, and optical clearing is applied to render retina transparent enabling the visualization of the peripheral retina and hyaloid vasculature in intact retina using light sheet fluorescent microscopy.

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole ...

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) imaging. They have received great attention in all research areas for the potential to analyze microscopic multicellular structures that extend over macroscopic distances. Given that kidney tubules, vasculature, nerves, and glomeruli extend in many directions, which have been only partially captured by traditional two-dimensional techniques so far, tissue clearing also opened up many new areas of kidney research. The list of optical clearing methods is rapidly growing, but it remains difficult for beginners in this field to choose the best method for a given research question. Provided here is a simple method that combines antibody labeling of thick mouse kidney slices; optical clearing with cheap, non-toxic and ready-to-use chemical ethyl cinnamate; and confocal imaging. This protocol describes how to perfuse kidneys and use an antigen-retrieval step to increase antibody- binding without requiring specialized equipment. Its application is presented in imaging different multicellular structures within the kidney, and how to troubleshoot poor antibody penetration into tissue is addressed. We also discuss the potential difficulties of imaging endogenous fluorophores and acquiring very large samples and how to overcome them. This simple protocol provides an easy-to-setup and comprehensive tool to study tissue in three dimensions.

The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) ...

Medicine

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the morphology of processes and substructures within these neurons. As such, much of what is known about brain function has arisen alongside developments in imaging technologies that allow further insight into how neurons are organized and connected in the brain. Improvements in tissue clearing have allowed for high-resolution imaging of thick brain slices, facilitating morphological reconstruction and analyses of neuronal substructures, such as dendritic arbors and spines. In tandem, advances in image processing software provide methods of quickly analyzing large imaging datasets. This work presents a relatively rapid method of processing, visualizing, and analyzing thick slices of labeled neural tissue at high-resolution using CLARITY tissue clearing, confocal microscopy, and image analysis. This protocol will facilitate efforts toward understanding the connectivity patterns and neuronal morphologies that characterize healthy brains, and the changes in these characteristics that arise in diseased brain states.

Neuronal dendritic morphology often underlies function. Indeed, many disease processes that affect the development of neurons manifest with a morphological phenotype. This protocol describes a simple and powerful method for analyzing intact dendritic arbors and their associated spines.

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the ...

Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain cells and their interactions. Viral vector transduction enables the marking of diverse cell types in different fluorescence colors within the same tissue. Cells can be identified genetically by activity or projection. Using a modified CLARITY protocol, the potential sample size of astrocytes and neurons has grown by 2-3 orders of magnitude. The use of CLARITY allows the imaging of complete astrocytes, which are too large to fit in their entirety in slices, and the examination of the somata with all their processes. In addition, it provides the opportunity to investigate the spatial interaction between astrocytes and different neuronal cell types, namely, the number of pyramidal neurons in each astrocytic domain or the proximity between astrocytes and specific inhibitory neuron populations. This paper describes, in detail, how these methods are to be applied.

Combining viral vector transduction and brain clearing using the CLARITY method allows the investigation of a large number of neurons and astrocytes simultaneously.

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain ...

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging from neural circuits to subcellular neuronal structures are visualized in mouse brain slices optically cleared with ScaleSF. This clearing method is a modified version of ScaleS and is a hydrophilic tissue clearing method for tissue slices that achieves potent clearing capability as well as a high-level of preservation of fluorescence signals and structural integrity. A customizable three dimensional (3D)-printed imaging chamber is designed for reliable mounting of cleared brain tissues. Mouse brains injected with an adeno-associated virus vector carrying enhanced green fluorescent protein gene were fixed with 4% paraformaldehyde and cut into slices of 1-mm thickness with a vibrating tissue slicer. The brain slices were cleared by following the clearing protocol, which include sequential incubations in three solutions, namely, ScaleS0 solution, phosphate buffer saline (&#8211;), and ScaleS4 solution, for a total of 10.5&#8211;14.5 h. The cleared brain slices were mounted on the imaging chamber and embedded in 1.5% agarose gel dissolved in ScaleS4D25(0) solution. The 3D image acquisition of the slices was carried out using a confocal laser scanning microscope equipped with a multi-immersion objective lens of a long working distance. Beginning with mesoscopic neuronal imaging, we succeeded in visualizing fine subcellular neuronal structures, such as dendritic spines and axonal boutons, in the optically cleared brain slices. This protocol would facilitate understanding of neuronal structures from circuit to subcellular component scales.

The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic levels.

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging ...

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

High-Resolution 3D Reconstruction of Human Brain with SHORT and LSFM

Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging

Despite the numerous clearing techniques that emerged in the last decade, processing postmortem human brains remains a challenging task due to its dimensions and complexity, which make imaging with micrometer resolution particularly difficult. This paper presents a protocol to perform the reconstruction of volumetric portions of the human brain by simultaneously processing tens of sections with the SHORT (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) tissue transformation protocol, which enables clearing, labeling, and sequential imaging of the samples with light-sheet fluorescence microscopy (LSFM). SHORT provides rapid tissue clearing and homogeneous multi-labeling of thick slices with several neuronal markers, enabling the identification of different neuronal subpopulations in both white and grey matter. After clearing, the slices are imaged via LSFM with micrometer resolution and in multiple channels simultaneously for a rapid 3D reconstruction. By combining SHORT with LSFM analysis within a routinely high-throughput protocol, it is possible to obtain the 3D cytoarchitecture reconstruction of large volumetric areas at high resolution in a short time, thus enabling comprehensive structural characterization of the human brain.

Author Spotlight: Advancing 3D Cytoarchitecture Analysis - Rapid Volumetric Reconstruction of the Human Brain 

The present protocol provides a step-by-step procedure for rapid and simultaneous optical clearing, muti-round labeling, and 3D volumetric reconstruction of tens of postmortem human brain sections by combining the (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) SHORT tissue transformation technique with light-sheet fluorescence microscopy imaging in a routinely high-throughput protocol.