Name: generation of three-dimensional printed biological innovative mold for optically transparent tissue specimens
Uploaded: 2018-01-16T08:48:05
Description: Histology continues to evolve in modern day experimentation. Recently, groups have been able to make tissue and bones transparent by removing lipids and ...

1. Clear the Tissue or Bone Specimens Following Established Clearing Methods
<ol>
	<li>Follow this protocol or another established tissue-clearing method: for passive CLARITY begin by making hydrogel monomer (HM) solution included of 1% (wt/vol) acrylamide, 0.0125 - 0.05% (wt/vol) bisacrylamide, 4% paraformaldehyde, 1x PBS, deionized water, and 0.25% of the thermal initiator into a 50-mL tube3.</li>
	<li>Perform a trans-cardial perfusion with HM and incubate the tissue overnight or 2 days at 4 &...

<ol>
	<li>Lagerweij, T., 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=Optical+clearing+and+fluorescence+deep-tissue+imaging+for+3D+quantitative+analysis+of+the+brain+tumor+microenvironment.">Optical clearing and fluorescence deep-tissue imaging for 3D quantitative analysis of the brain tumor microenvironment.</a> Angiogenesis. 20, 533-546 (2017).</li><li>Miller, S. 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=3D+Printer+Generated+Tissue+iMolds+for+Cleared+Tissue+Using+Single-+and+Multi-Photon+Microscopy+for+Deep+Tissue+Evaluation.">3D Printer Generated Tissue iMolds for Cleared Tissue Using Single- and Multi-Photon Microscopy for Deep Tissue Evaluation.</a> Biol Proced Online. 19, 7 (2017).</li><li>Tomer, R., Ye, L., Hsueh, B., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+CLARITY+for+rapid+and+high-resolution+imaging+of+intact+tissues.">Advanced CLARITY for rapid and high-resolution imaging of intact tissues.</a> Nat Protoc. 9, 1682-1697 (2014).</li><li>Yang, Y., 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=Molecular+comparison+of+GLT1++and+ALDH1L1++astrocytes+in+vivo+in+astroglial+reporter+mice.">Molecular comparison of GLT1+ and ALDH1L1+ astrocytes in vivo in astroglial reporter mice.</a> Glia. 59, 200-207 (2011).</li><li>Miller, S. 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=Astroglia+in+Thick+Tissue+with+Super+Resolution+and+Cellular+Reconstruction.">Astroglia in Thick Tissue with Super Resolution and Cellular Reconstruction.</a> PLoS One. 11, 0160391 (2016).</li><li>Theis, M., Giaume, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin-based+intercellular+communication+and+astrocyte+heterogeneity.">Connexin-based intercellular communication and astrocyte heterogeneity.</a> Brain Res. 1487, 88-98 (2012).</li><li>Cabezas, R., 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=Astrocytic+modulation+of+blood+brain+barrier:+perspectives+on+Parkinson's+disease.">Astrocytic modulation of blood brain barrier: perspectives on Parkinson's disease.</a> Front Cell Neurosci. 8, 211 (2014).</li></ol>

Optical clearing methods are continuing to increase the understanding of environmental niches and cellular heterogeneity. The tissue is maintained and stored in a hydrogel complex that allows biomacromolecules to permeate the matrix. This allows for groups to study immunofluorescence of proteins and nucleic acids either by endogenous fluorescence or post-hoc labeling, across large volumes of tissue.
However, a major caveat in the system comes when attempting to image the tissue. In current met...

Whole tissue clearing methods have recently become a widely-used method in histology. First the tissue and organ are cleared resulting in the specimen becoming optically-transparent, then this is followed by staining and imaging. With these techniques scientists are able to image at high resolution entire organs and thick specimens. In addition, many tissue clearing methodologies allow for repeated staining. This provides users the ability to develop 3D renderings of structural and functional relationships across a large volume of area.
The 3D renderings that are produced have application in many fields of science. For example, scientists can now clear and generate a 3D model of a tumor at cellular and subcellular resolution1. This allows scientists the ability to study the heterogeneity of cells, create 3D renderings of entire organs to examine cell interactions, vascularization, and environment niches. Furthermore, using systems like this also allows a whole organ evaluation after drug screenings and other therapeutic interventions. The benefits of tissue clearing make it imperative that there is a rapid and reliable method to structurally support any specimen being imaged to the micron level and also provide a method to place multiple organs on one imaging platform, to allow for screening of multiple specimens.
For these reasons, the tissue iMold has been generated2. iMolds are made to be utilized by researchers evaluating large tissue specimens that are optically transparent. The iMold restricts all tissue movement during prolonged and repeated imaging. Current methodologies lead to specimen movement during imaging and re-imaging3. In addition, current methods make it near impossible for sequential staining of the same cellular environment, due to relocating precise anatomical locations. These caveats are resolved with the generation of the tissue specific iMolds.
iMolds can be generated by almost all 3D printers and used by any research laboratory. First, they are generated at the micron level with the material of choice, such as PLA plastic. Then the iMold is mounted and can be used for repeated imaging. iMolds also allow the user to image multiple organs on one imaging platform during one imaging session, or repeated sessions. They are inexpensive to generate and reliable to use. The development of the iMold and representative results are detailed in this protocol.

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			<td height="21" style="height:21px;width:216px;">Acrylamide</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:119px;">A9099</td>
			<td style="width:303px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bisacrylamide</td>
			<td style="width:101px;">VWR</td>
			<td style="width:119px;">0172-50G</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">paraformaldehyde</td>
			<td style="width:101px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15714-S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PBS</td>
			<td style="width:101px;">Quality Biological</td>
			<td style="width:119px;">119-069-131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">thermal initiator&#160;</td>
			<td>Wako Chemicals</td>
			<td style="width:119px;">VA-044&#160;</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">sodium dodecyl sulfate&#160;</td>
			<td style="width:101px;">Bio-Rad</td>
			<td style="width:119px;">161-0302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">boric acid&#160;</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:119px;">B-7660</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:119px;">X100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">focus clear</td>
			<td style="width:101px;">CEDARLANE</td>
			<td>F101-KIT</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Google Sketchup</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Camera</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td>we used an iPhone 7 camera</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">3D Printer</td>
			<td style="width:101px;">M3D</td>
			<td style="width:119px;">Micro +</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PLA plastic roll for 3D Printer</td>
			<td style="width:101px;">M3D</td>
			<td style="width:119px;">3D Ink&#160;</td>
			<td>no catalog number listed, we use black PLA filament</td>
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	</tbody>
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generation of three-dimensional printed biological innovative mold for optically transparent tissue specimens

Histology continues to evolve in modern day experimentation. Recently, groups have been able to make tissue and bones transparent by removing lipids and other biological species that prevent deep tissue imaging. Now, groups can robustly "clear" the tissue or specimen and perform a wide array of microscopy such as single-photon, multiphoton, and light-sheet to evaluate their biological target of interest. This has eliminated the need to freeze, cyro-slice, and cyro-preserve tissue, greatly limiting human prone error. However, current tissue clearing methods still lack a robust system to mount and structurally support unique specimens. This leads to issues with tissue movement during imaging, the lack of reliability when re-staining and re-evaluating specimens, and working with abnormally shaped specimens such as tumors. To overcome these obstacles, the innovative Mold (iMold) was generated. iMolds are first created from images of the specimen followed by three-dimensional (3D) printed molds that support the unique tissue structure being imaged and re-imaged. This leads to a dramatic reduction in tissue movement while imaging and allows for scientists to robustly re-evaluate areas of interest that have been re-stained or need to be re-imaged. Furthermore, iMolds can be created in a short time period and generated for multiple samples in any imaging format (i.e. slide, cell culture plate). This allows users to scan multiple organs or specimens on one slide or cell culture dish. In closing, iMolds can be utilized by any research laboratory studying cleared specimens while also reducing costs, time, and tissue movement compared to other current methodologies.

The benefits of this method, in combination with tissue clearing methods is the ability to control movement of tissue to avoid shifting during long term imaging and/or the re-staining of tissue. Therefore, it is critical that the user designs a properly fitted iMold, have proper tissue-clearing, and have the validation of the immunofluorescent target (i.e. RNA, Protein). Other methods include using putty or iSpacers to maintain the tissue, however this leads to the issues previou...

Tissue innovative Molds (iMolds) have been developed to reduce specimen movement, structurally support the specimen being imaged, and allow for repeated imaging on precise anatomical locations using optically-transparent samples.

Generation of Three-dimensional Printed Biological Innovative Mold for Optically Transparent Tissue Specimens

Histology continues to evolve in modern day experimentation. Recently, groups have been able to make tissue and bones transparent by removing lipids and ...