Name: 3d printing of preclinical x-ray computed tomographic data sets
Uploaded: 2013-03-22T13:00:00
Duration: 11 min 6 s
Description: Watch this Scientific Journal Video about 3D Printing of Preclinical X-ray Computed Tomographic Data Sets at JoVE.com

We warmly thank the Nanovic Institute for European Studies, the Glynn Family Honors Program, Notre Dame Integrated Imaging Facility (NDIIF) and Carestream Health for financial support for this project. Research on rabbit cranial development supported by NSF BCS-1029149 to MJR.

1. Animals
<ol>
 <li>For the results reported below, one male Lobund-Wistar rat of ten months of age was obtained from the Freimann Life Science Center, University of Notre Dame (Notre Dame, Indiana, USA). An ex vivo New Zealand White Rabbit (Male, age = 8 weeks) skull sample, preserved in 10% formalin, was obtained from the laboratory of Prof. Matthew Ravosa, University of Notre Dame. </li>
 <li>For in vivo imaging, the rat was anesthetized by Isofluorane (2.5% flow rate) with maintenance via a nose-cone system. The animal was positioned prone in the standard rat bed (M2M Imaging Inc., Cleveland, Ohio) supplied with the Albira image station. Limbs were positioned lateral from the torso for a uniform CT acquisition. </li>
 <li>After image acquisition was completed, the rat was removed from the nose cone and returned to the recovery cage until ambulatory. </li>
 <li>For scans of the rabbit skull, the specimen was placed in the rat bed in a sealed plastic bag containing formalin. </li>
</ol>
2. Image Acquisition and Reconstruction
<ol>
 <li>In Vivo and Ex Vivo Image acquisitions were performed using the Albira CT system (Carestream Molecular Imaging, Woodbridge, CT). The system was set to scan a bed of 180 mm length by performing three circular scans (600 projections per scan), each with a 65 mm FOV, which are then stitched together during reconstruction. The X-ray source was set to a current of 400 &mu;A and voltage of 45 kVp, and used a 0.5 mm Al filter to harden the beam. Approximate radiation deep dose equivalent for CT settings was 660 mSv, and shallow dose equivalent was 1171 mSv. These doses are over 10-fold lower than reported LD50 values. </li>
 <li>Images are reconstructed using the FBP (Filtered Back Projection) algorithm via the Albira Suite 5.0 Reconstructor using "Standard" parameters. These combined acquisition and reconstruction settings produce a final image with .125 mm isotropic voxels, deemed sufficient for whole animal analysis and 3D printing of anatomical structures. </li>
</ol>
3. Data Processing
<ol>
 <li>Skeletal features from CT scans may be printed from the raw data without segmentation. However, segmentation of soft tissues is required prior to processing the data for 3D printing. Here we show an example with lung tissue.
 <ol>
 <li>Open original MicroPET (data format for all modalities on the Albira Imaging System) file
 </li>
 <li>Draw a volume of interest (VOI) around the mouse so that all external space is removed.
 </li>
 <li>Under the 'VOI Tools' tab select 'Mask Outside Selected VOI' and set the masking value to -1,000 Hounsfield Units (HU, radiodensity scale for CT), which will effectively set the external space to the CT density value of air.</li>
 <li>With all the external space removed, under the 'Tools' tab, select 'External,' then click drop down arrow and select 'Segmentation'
 </li>
 <li>Set range to -550 to -200 and click 'Ok.'
 </li>
 <li>If file is very large, select 'Tools' tab, select 'Reduce,' and run the program
 </li>
 <li>Save as an Analyze file </li>
 </ol>
 </li>
 <li>Data must first be converted into DICOM format using PMOD (PMOD Technologies LTD, Zurich, Switzerland) analysis software. 
 <ol>
 <li>Open the PMOD Image Processing Software Package. </li>
 <li>Along the top row, select the View tab.</li>
 <li>In the lower right hand toolbar, click the downward arrow labeled Database under Load. </li>
 <li>Select MicroPET for the raw CT data, or Analyze for the segmented lung. </li>
 <li>Select the appropriate file and click Add to Selected.</li>
 <li>Click Open. </li>
 <li>In the lower right hand toolbar, click the upward arrow under Save. </li>
 <li>From this menu, choose the file type DICOM. </li>
 <li>Name the file and select Save. </li>
 <li>Close PMOD. </li>
 </ol>
 </li>
 <li>DICOM data contains volumetric density values for each voxel. In order to print this data, it must be processed as a contiguous surface instead of a volume. ImageJ v1.43u will be used to obtain surface renderings for further processing.
 <ol>
 <li>Open ImageJ Image Processing Software
 </li>
 <li>Select File &gt; Import. </li>
 <li>Select Image Sequence.</li>
 <li>Navigate to the file containing the newly created DICOM and select it.
 </li>
 <li>Select Plug-ins &gt; 3D &gt; 3D Viewer. </li>
 <li>Two windows will appear, the 3D viewer will appear and an ADD window. </li>
 <li>Under Display as in the ADD window change Volume to Surface.</li>
 <li>Change the default value of Threshold to 210. </li>
 <li>Click OK. </li>
 <li>In the 3D Viewer menu bar, select File &gt; Export surfaces as &gt; WaveFront. </li>
 <li>Name the file and click Save. </li>
 </ol>
 </li>
 <li>Two programs, Meshlab v1.3.1 and Netfabb Studio Basic 4.9, will concurrently remove any excess mesh, join together disconnected meshes, repair holes, and smooth the final mesh. The primary differences between these two programs are the tool sets made available to the user, and some of the interface navigation control. They are both 3D mesh editing software programs, and their use together provides the easiest approach to editing the model. 
<img alt="Icon 1" fo:src="/files/ftp_upload/50250/50250icon1highres.jpg" src="/files/ftp_upload/50250/50250icon1.jpg" /> indicates that these actions should be executed in Meshlab v1.3.1 
<img alt="Icon 2" fo:src="/files/ftp_upload/50250/50250icon2highres.jpg" src="/files/ftp_upload/50250/50250icon2.jpg" /> indicates that these actions should be executed in Netfabb Studio Basic 4.9
<ol>
 <li>To import meshes into editing software:
 <ol>
 <li>Open Meshlab v1.3.1 <img alt="Icon 1" fo:src="/files/ftp_upload/50250/50250icon1highres.jpg" src="/files/ftp_upload/50250/50250icon1.jpg" /></li>
 <li>From the menu bar select File &gt; New Empty Project.</li>
 <li>Selct File &gt; Import Mesh.</li>
 <li>Select the file and click Open.</li>
 <li>Once the file has loaded, a dialogue box will open. Keeping Unify Duplicated Vertices checked, press OK.
<ol>
 <li>Open Netfabb Studio Basic 4.9. <img alt="Icon 2" fo:src="/files/ftp_upload/50250/50250icon2highres.jpg" src="/files/ftp_upload/50250/50250icon2.jpg" /> </li>
 <li>Drag the desired file straight onto the Netfabb Studio Basic screen. </li>
 </ol>
 </li>
 </ol>
 </li>
 <li>To remove unwanted mesh from the surface :
 <ol>
 <li>From the menu bar in Meshlab, select Filters &gt; Cleaning and Repairing &gt; Remove Isolated Pieces (WRT Diameter). <img alt="Icon 1" fo:src="/files/ftp_upload/50250/50250icon1highres.jpg" src="/files/ftp_upload/50250/50250icon1.jpg" /></li>
 <li>Remove isolated connected components whose diameter is smaller than the specified constant. Enter a maximum diameter for these components and click Apply. </li>
 <li>Gradually, increase the maximum diameter to remove bigger pieces. Click Apply after each diameter modification. </li>
 </ol>
 </li>
 <li>To join disconnected pieces, holes must be cut in the current meshes at the desired locations of a new bond, and a bridge of mesh must be built between.
 <ol>
 <li>Use the Selection Tool, seventh tool from the left in the menu bar, to select the pieces of mesh that will be cut out. <img alt="Icon 1" fo:src="/files/ftp_upload/50250/50250icon1highres.jpg" src="/files/ftp_upload/50250/50250icon1.jpg" /></li>
 <li>Delete selected pieces of mesh with the Delete Faces button, the third tool from the left in the menu bar. </li>
 <li>From the menu bar, select File &gt; Export Mesh As.</li>
 <li>Name the file and change the file type to STL. </li>
 <li>Press Save. </li>
 <li>A dialogue box with saving options will appear, press OK. <img alt="Icon 2" fo:src="/files/ftp_upload/50250/50250icon2highres.jpg" src="/files/ftp_upload/50250/50250icon2.jpg" /> </li>
 <li>Drag this new file into Netfabb.</li>
 <li>On the top menu, select Repair, the fourth tool from the left. </li>
 <li>Select the Add triangles tool, thirteenth from the left. </li>
 <li>Click on an open edge (they will be yellow) on one side and then click on an open edge on the other piece. Create 5-10 bridges across the gap. </li>
 <li>Select the Automatic Repair button at the bottom right </li>
 <li>Highlight Default Repair. </li>
 <li>Click Execute. </li>
 <li>Press Cancel after the repair has been executed. </li>
 </ol>
 Optional: Some created triangles may be the wrong orientation. The following steps can be used to reorient these triangles.
 <ol>
 <li> Select the Flip Selected Triangles Button. </li>
 <li> Click on any disoriented triangles to rotate all triangles in the correct direction. </li>
 </ol>
 </li>
 <li>To repair craters and holes, entire holes and craters must be removed and bridges must be built on each side of the mesh.
 <ol>
 <li>Click the Selection Tool. <img alt="Icon 1" fo:src="/files/ftp_upload/50250/50250icon1highres.jpg" src="/files/ftp_upload/50250/50250icon1.jpg" /></li>
 <li>Select the crater or hole to be filled. </li>
 <li>Click the Delete Faces button.</li>
 <li>From the menu bar, select File &gt; Export Mesh As.</li>
 <li>Name the file and change the file type to STL. </li>
 <li>Press Save. </li>
 <li>A dialogue box with saving options will appear, press OK. </li>
 <li>Drag this new file into Netfabb. <img alt="Icon 2" fo:src="/files/ftp_upload/50250/50250icon2highres.jpg" src="/files/ftp_upload/50250/50250icon2.jpg" /> </li>
 <li>On the top menu, select Repair, the fourth tool from the left.</li>
 <li>Select the Add triangles tool, thirteenth from the left. </li>
 <li>Click on an open edge (they will be yellow) on one side and then click on an open edge on the other piece. Create 5-10 bridges across the gap. </li>
 <li>Select the Automatic Repair button at the bottom right </li>
 <li>Highlight Default Repair. </li>
 <li>Click Execute. </li>
 <li>Press Cancel after the repair has been executed. </li>
 </ol>
 </li>
 <li>Laplacian smoothing, the smoothing algorithm in Meshlab, is used to smooth the object while still preserving the structural integrity of the model. 
 <ol>
 <li>Navigate Filters &gt; Smoothing &gt; Fairing and Deformation &gt; Laplacian Smooth . <img alt="Icon 1" fo:src="/files/ftp_upload/50250/50250icon1highres.jpg" src="/files/ftp_upload/50250/50250icon1.jpg" /> </li>
 <li>Select a number of smoothing iterations. More iterations will result in a smoother model, but each iteration slowly degrades the volume of the model, which can result in disconnected pieces and sharp edges. 1-5 iterations are recommended. </li>
 <li>Press OK. </li>
 <li>From the menu bar, select File &gt; Export Mesh As. </li>
 <li>Name the file and change the file type to STL.</li>
 <li>Press Save.</li>
 <li>A dialogue box with saving options will appear, press OK. </li>
 <li>Drag this new file into Netfabb. <img alt="Icon 2" fo:src="/files/ftp_upload/50250/50250icon2highres.jpg" src="/files/ftp_upload/50250/50250icon2.jpg" /> </li>
 <li>On the top menu, select Repair, the fourth tool from the left.</li>
 <li>Select the Add triangles tool, thirteenth from the left. </li>
 <li>Click on an open edge (they will be yellow) on one side and then click on an open edge on the other piece. Create 5-10 bridges across the gap. </li>
 <li>Select the Automatic Repair button at the bottom right </li>
 <li>Highlight Default Repair. </li>
 <li>Click Execute. </li>
 <li>Press Cancel after the repair has been executed. </li>
 <li>From the menu bar, select File &gt; Export Mesh As.</li>
 <li>Name the final file and change the file type to STL. </li>
 <li>Press Save. </li>
 <li>A dialogue box with saving options will appear, press OK. </li>
 </ol>
 </li>
 </ol>
 </li>
</ol>
4. Printing
<ol>
 <li>Printing using the Makerbot
 <ol>
 <li>Open the STL file in ReplicatorG. ReplicatorG is a Makerbot Industries program used to communicate with the Makerbot. </li>
 <li>Click Scale from the lower right hand corner menu and select Fill the Build Space!</li>
 <li>Select Rotate and click Lay Flat. </li>
 <li>Click Center. </li>
 <li>For models with fine detail, select fill build platform to scale up model. </li>
 <li>From the same menu, click Move and select Put on Platform. </li>
 <li>Once the proper orientation has been achieved, select Generate GCODE from the top menu bar. 
 *A window with printing options will appear. </li>
 <li>Select the extruder that will provide the filament to print the object (Left or Right).</li>
 <li>Select Use Raft/Support. </li>
 <li>From the drop down menu entitled Use support material, select Full Support. </li>
 <li>Select Generate GCODE. A pop-up box showing the progress of the GCODE will appear. </li>
 <li>Once the GCODE has been completed, select Build to file for use with SD card.</li>
 <li>Click Save. </li>
 <li>Drag the file onto an SD card. </li>
 <li>Place the SD card into the Makerbot and using the Makerbot keypad select Print from SD. </li>
 <li>Under Print from SD, select the desired file name. The Makerbot will automatically begin to warm-up to print the object. </li>
 </ol>
 </li>
 <li>Shapeways Printing 
 <ol>
 <li>After creating a free account with Shapeways, the STL file can be directly uploaded to the Shapeways website: <a href="http://www.shapeways.com/upload/" target="_blank">http://www.shapeways.com/upload/</a></li>
 <li>Click Upload and select the STL file. </li>
 <li>Select a Title for the uploaded file. </li>
 <li>Select a Unit of Measure from the dropdown menu. </li>
 <li>Click Upload Model. </li>
 <li>The file is now ready for printing through Shapeways. Once the file is uploaded Shapeways will take a few minutes to process the file to be sure it can actually be printed. You will be able to print the model from the "my models" page after about ten minutes.</li>
 <li>The "white strong flexible" selection was used to print skeletal structures, while "purple strong flexible" was used for lung tissue.</li>
 </ol>
 </li>
 <li>ProJet HD 3000 Printing 
 <ol>
 <li>The STL file may also be printed out using a commercial high-resolution three-dimensional printer like the ProJet HD 3000 (Springboard Engineering Solutions LLC, Innovation Park, Notre Dame, IN, USA). </li>
 <li>The STL file is loaded into the 3D Systems proprietary software to layout the job on the platform. This requires changing the orientation of the model around to minimize the use of wax support and print time. This file is saved. </li>
 <li>The job is then electronically sent to the printer.</li>
 <li>A platform of aluminum is loaded into the printer and the ProJet HD 3000 begins to print the object. </li>
 <li>The model is then removed from the platform and placed into an oven at approximately 73 &deg;C to melt the support wax from the model. </li>
 <li>The object is removed hot and wiped down with a Kimwipe to remove remaining surface wax.</li>
 </ol>
 </li>
</ol>

<ol>
	<li>Kazmer, D. O., Speight, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+Injection+Molding+Technology+for+the+Next+Millennium.">Polymer Injection Molding Technology for the Next Millennium.</a> Journal of Injection Molding Technology. 1 (2), 81-90 (1997).</li><li>Bradshaw, S., Bowyer, A., Haufe, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intellectual+property+implications+of+low-cost+3D+printing.">The intellectual property implications of low-cost 3D printing.</a> ScriptEd. 7 (1), 5-31 (2010).</li><li>Partridge, R., Conlisk, N., Davies, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-lab+three-dimensional+printing:+An+inexpensive+tool+for+experimentation+and+visualization+for+the+field+of+organogenesis.">In-lab three-dimensional printing: An inexpensive tool for experimentation and visualization for the field of organogenesis.</a> Organogenesis. 8 (1), 1-6 (2012).</li><li>Guillot, A., Champely, S., Batier, C., Thiriet, P., Collet, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+spatial+abilities,+mental+rotation+and+functional+anatomy+learning.">Relationship between spatial abilities, mental rotation and functional anatomy learning.</a> Adv. Health Sci. Educ. Theory Pract. 12, 491-507 (2007).</li><li>Cignoni, P., Scopigno, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sampled+3D+models+for+CH+applications:+a+viable+and+enabling+new+medium+or+just+a+technological+exercise.">Sampled 3D models for CH applications: a viable and enabling new medium or just a technological exercise.</a> Association for Computing Machinery Journal on Computing and Cultural. 1 (1), 1 (2008).</li><li>Symes, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+3D-printed+reactionware+for+chemical+synthesis+and+analysis.">Integrated 3D-printed reactionware for chemical synthesis and analysis.</a> Nature Chemistry. 4, 349-354 (2012).</li><li>Sheridan, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabricating+consent:+three-dimensional+objects+as+rhetorical+compositions.">Fabricating consent: three-dimensional objects as rhetorical compositions.</a> Computers and Composition. 27, 249-265 (2010).</li><li>Windisch, G., Salaberger, D., Rosmarin, W., Kastner, J., Exner, G. U., Haldi-Brandle, V., Anderhuber, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Model+for+Clubfood+Based+on+Micro-CT.">A Model for Clubfood Based on Micro-CT.</a> Data. J. Anat. 210, 761-766 (2007).</li></ol>

W. Matthew Leevy is a consultant for Carestream Molecular Imaging. Brian Stamile is a support engineer for MakerBot Industries.

X-ray CT data sets of a living Lobund-Wistar rat and an ex vivo New Zealand White Rabbit skull were utilized to demonstrate the feasibility of 3D object production from pre-clinical biological data. Models were generated using three difference sources: 1)The popular Makerbot Replicator, 2) The third party company Shapeways Inc, and 3) The high-grade commercial ProJet HD 3000. Each printer was able to generate objects that satisfied the principle goal of enhanced data visualization. 
During the process of printing pre-clinical CT data, the advantages and disadvantages of each method of printing were ascertained and summarized for the end user. The MakerBot Replicator is an inexpensive ($1,750) bench top solution that is accessible to virtually any lab around the globe. It can print in multiple colors with inexpensive inputs (a rat CT with lungs used about $3.50 in plastic). However, the Makerbot is limited by resolution, and thus some models will have to be enlarged for proper extrusion and visualization of intended structure. Shapeways Inc. provides an outstanding number of selections with regard to color and material. The models are high resolution, and robust. While their prices are about 10-fold higher than the MakerBot on a per unit basis (a rat CT with lungs was $41.61), a user can execute a limited number of jobs and avoid the upfront cost of purchasing a printer. The two-week lead time from Shapeways is a minor disadvantage. The ProJet HD 3000 provided outstanding models in terms of resolution and strength. We were fortunate enough to contract the printing of our objects on the ProJet HD 3000 at Innovation Park at Notre Dame (about $30 for a rat CT with lungs for labor and materials). Users may have difficulty with access to this type of equipment as they are priced in the range of $80,000, and it is cumbersome to print with multiple colors as well. Since each instrument/manufacturer provides a different metric to describe the resolution for object printing (Shapeways minimum level of detail = 0.2 mm, minimum wall thickness = 0.7 mm, 5 MakerBot slice thickness = 0.2-0.3 mm with a 0.4 mm nozzle, 6ProJet HD 3000 DPI = 656 x 656 x 800 with an accuracy of 0.025-0.05 mm ), a qualitative assessment of relative resolutions between each system suggests that both Shapeways and the ProJet HD system can print in high detail to scale, while some objects must be enlarged for successful use of the MakerBot. Collectively, all three methods are environmentally friendly and provide a convenient means to achieve facile production of highly detailed pre-clinical X-ray CT models. 
Conclusion
Gradually, the technology of 3D printing has become more accessible as both costs and complexity have been minimized.8, 9 Now, literally anyone may print high-resolution, three-dimensional objects from digital files. These detailed three-dimensional objects can be useful tools for both educators and researchers alike. Furthermore, they provide a means of visual communication that assists in achieving a clearer understanding.10 For example, medical researchers can use specimen or patient-specific models to improve both communication and comprehension with their colleagues and patients.11 Although representation on 2D screens has come a long way, there is absolutely no replacement for the visual and sensory experience of holding a real object that is able to be held, rotated, examined and moved around. A model paired with an electronic data representation is even more powerful as it allows researchers to examine the physical object for regions of interest, and to find those areas on a computer model for further quantitative analysis. With proper data collection, surface rendering, and stereolithographic editing, it is possible to rapidly produce detailed, relatively inexpensive models from X-ray CT data. Here, we provide a detailed, step by step method for the production of a three-dimensional model from pre-clinical small animal data collected with an X-ray micro-CT. We acquired our in vivo and ex vivo CT data sets using an Albira image station, and performed subsequent processing with PMOD, ImageJ, Meshlab and Netfabb software packages. Finally, we provide detailed instructions to enable three-dimensional model printing with a range of commercial solutions. In each case, the end result is a model that provides a unique, hand-held, physical manifestation of the acquired tomographic data that would normally be restricted to a computer screen.

<table border="1">
	<tbody>
 	<tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>
Required Programs			
<ol>
	<li>Albira Image Acquirer </li>
 <li>PMOD </li>
 <li>ImageJ </li>
 <li>Meshlab </li>
 <li>Netfabb </li>
 <li>ReplicatorG </li>
</ol>
</td>
</tr>
</tbody>
</table>

3d printing of preclinical x-ray computed tomographic data sets

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing. Traditional, mold-injection methods to create models or parts have several limitations, the most important of which is a difficulty in making highly complex products in a timely, cost-effective manner.1 However, gradual improvements in three-dimensional printing technology have resulted in both high-end and economy instruments that are now available for the facile production of customized models.2 These printers have the ability to extrude high-resolution objects with enough detail to accurately represent in vivo images generated from a preclinical X-ray CT scanner. With proper data collection, surface rendering, and stereolithographic editing, it is now possible and inexpensive to rapidly produce detailed skeletal and soft tissue structures from X-ray CT data. Even in the early stages of development, the anatomical models produced by three-dimensional printing appeal to both educators and researchers who can utilize the technology to improve visualization proficiency. 3, 4 The real benefits of this method result from the tangible experience a researcher can have with data that cannot be adequately conveyed through a computer screen. The translation of pre-clinical 3D data to a physical object that is an exact copy of the test subject is a powerful tool for visualization and communication, especially for relating imaging research to students, or those in other fields. Here, we provide a detailed method for printing plastic models of bone and organ structures derived from X-ray CT scans utilizing an Albira X-ray CT system in conjunction with PMOD, ImageJ, Meshlab, Netfabb, and ReplicatorG software packages.

<img alt="Figure 1" src="/files/ftp_upload/50250/50250fig1.jpg" /> 
Figure 1. 3D printed models of the lungs and skeletal features of a rat X-ray CT data set. Objects were printed using a ProJet HD 3000 (left), Shapeways Inc. (Center) or a Makerbot Replicator (right). The scale bar denotes 2 cm. Note that the scale bar in panel C is smaller than that of A and B, which reflects that in some cases the Makerbot must print an enlarged object in order to output sufficient detail.
Figure 1 depicts the final products for three methods of printing of the same in vivo rat CT data set. All three models consist of a cropped skeletal structure and removable lungs which were printed independently and pieced together. The model at left is the result of the ProJet HD 3000 high-resolution printer, created using translucent acrylic plastic. The object at center was produced using a third party company, Shapeways Inc., in which the skeletal structure was printed using nylon 12 white plastic while the respiratory structures were fabricated in purple. These first two models were printed to actual scale, measuring approximately 11 cm in length. The object at right was made using the MakerBot. The skeletal structure was printed using natural colored ABS (acrylonitrile butadiene styrene) plastic and the lungs with lime green ABS. Because of the resolution limits of the MakerBot, this model could not be printed to scale without degradation of fine structure like the rib cage. Instead, the model was scaled up by almost 2X using the "fill build space" option to obtain the desired visual detail, resulting in an object of 21 cm in length. 
<img alt="Figure 2" src="/files/ftp_upload/50250/50250fig2.jpg" /> 
Figure 2. 3D printed models of a an ex vivo rabbit skull data set. The objects displayed were printed using a ProJet HD 3000 (left), Shapeways Inc. (Center) and a Makerbot Replicator (right). The scale bar denotes 1 cm.
Figure 2 shows the final products of each method of printing for the ex vivo rabbit skull CT data set. The model at left is the result from the ProJet HD 3000 high-resolution printer using translucent acrylic plastic. The model at center was printed in white nylon12 plastic through Shapeways Printing. The object at right was printed in white plastic using the MakerBot. All three objects were printed to scale and measure approximately 8.5 cm in length. 
<img alt="Figure 3" src="/files/ftp_upload/50250/50250fig3.jpg" /> 
Figure 3. 3D printed models of a full rat X-ray CT data set. Objects were printed using a ProJet HD 3000 (left), and Shapeways Inc. (right). The scale bar denotes 1 cm.
 Figure 3 depicts the final products for two methods of printing of a full in vivo CT data set of a rat. Both models consist of a complete skeletal structure (minus the tail) and removable lungs. The model at left is the resultant of a high-resolution printer, the ProJet HD 3000, printed using translucent acrylic plastic. The model on the right was printed using Shapeways Printing, with the skeletal structure created using white nylon12 plastic and the lungs in purple. These two models were printed to actual scale, measuring approximately 19 cm in length. Because of the intricate detail required, the full skeleton could not be printed with the MakerBot Replicator. 
 During the exploration of three-dimensional printing techniques, certain advantages and disadvantages were observed and are outlined in Table 1.
<table border="1">
<tbody>
 <tr>
 <td>Method of Printing</td>
 <td>Advantages</td>
 <td>Disadvantages</td>
 </tr>
 <tr>
 <td>MakerBot</td>
 <td>Extremely fast, variety of color options, able to print in two colors, extremely inexpensive</td>
 <td>Lowest level of detail. Removal of support materials is slow (on the order of a couple hours).</td>
 </tr>
 <tr>
 <td>Shapeways</td>
 <td>Varity of color options, variety of materials for printing, high level of detail, relatively inexpensive</td>
 <td>Two-week time to process and receive an order</td>
 </tr>
 <tr>
 <td>ProJet HD 3000</td>
 <td>Relatively quick turnaround, highest level of detail, high throughput, easy to remove support materials (wax).</td>
 <td>Most expensive up front cost, only one color option during practical use.</td>
 </tr>
 </tbody>
</table>
Table 1. Comparison of 3D printing technologies available to print CT data sets.

Watch this Scientific Journal Video about 3D Printing of Preclinical X-ray Computed Tomographic Data Sets at JoVE.com

3D Printing of Preclinical X-ray Computed Tomographic Data Sets

Using modern plastic extrusion and printing technologies, it is now possible to quickly and inexpensively produce physical models of X-ray CT data taken in a laboratory. The three -dimensional printing of tomographic data is a powerful visualization, research, and educational tool that may now be accessed by the preclinical imaging community.

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing.  Traditional, ...

3d-printing-of-preclinical-x-ray-computed-tomographic-data-sets

Research

JoVE Journal

Biology

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

Microcontact Printing of Proteins for Cell Biology

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. Development of microcontact printing by the Whitesides group (<a href="http://gmwgroup.harvard.edu/">http://gmwgroup.harvard.edu/</a>) in the mid-1990s revolutionalized this field by making microelectronics/microfabrication techniques accessible to laboratories focused on the life sciences. Initial implementations of this method used polydimethylsiloxane (PDMS) stamps to create patterns of functionalized chemicals on material surfaces1. Since then, a range of innovative approaches have been developed to pattern other molecules, including proteins2. This video demonstrates the basic process of creating PDMS stamps and uses them to pattern proteins, as these steps are difficult to accurately express in words. We focus on patterning the extracellular matrix protein fibronectin onto glass coverslips as a specific example of patterning.
An important component of the microcontact printing process is a topological master, from which the stamps are cast; the raised and lowered regions of the master are mirrored into the stamp and define the final pattern. Typically, a master consists of a silicon wafer coated with photoresist and then patterned by photolithography, as is done here. Creation of masters containing a specific pattern requires specialized equipment, and is best approached in consultation with a fabrication center or facility. However, almost any substrate with topology can be used as a master, such as plastic diffraction gratings (see Reagents for one example), and such serendipitous masters provide readily available, simple patterns. This protocol begins at the point of having a master in hand.

Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces. We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass.

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. ...

Protein Crystallization for X-ray Crystallography

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.
To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).
Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.
Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.

The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography.

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with single-particle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules. 
The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules. 
The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level.

This paper describes the cryo-electron microscopy methodology, which is used to obtain high quality microscopic images of macromolecules in their near-native state. The method yields images suitable for further computerized processing using the single particle approach, devised to generate the 3D reconstruction of a macromolecule.

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state ...

SpOT the Correct Tissue Every Time in Multi-tissue Blocks

Multi-tissue paraffin blocks provide high throughput analysis with increased efficiency, experimental uniformity, and reduced time and cost. Tissue microarrays make up the majority of multi-tissue paraffin blocks, but increasingly, researchers are using non-arrayed blocks containing larger tissues from multiple individuals which can provide many of the advantages of tissue microarrays without substantial investment in planning and equipment. A critical component of any multi-tissue analysis is the orientation method used to identify each individual tissue. Although methods exist to maintain proper orientation and identification of tissues in multi-tissue blocks, most are not well-suited to non-arrayed blocks, may consume valuable space within an array and/or are difficult to produce in the standard histology laboratory. The Specimen Orientation Tag (SpOT) is a simple, low cost orientation tool that is clearly visible in paraffin blocks and all tissue sections for reliable specimen identification in arrayed and non-arrayed layouts. The SpOT provides advantages over existing orientation methods for non-arrayed blocks as it does not require any direct modification to the tissue and allows for flexibility in the arrangement of tissue pieces.

The purpose of the Specimen Orientation Tag (SpOT) is to function as an orientation tool to aid in individual tissue identification in multi-tissue paraffin blocks. These protocols demonstrate how it is constructed easily from common, low-cost histology materials and serves as a reliable visual marker in paraffin blocks and sections.

Multi-tissue paraffin blocks provide high throughput analysis with increased efficiency, experimental uniformity, and reduced time and cost. Tissue ...

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. The S domain forms a contiguous scaffold around the viral RNA, whereas the P domain forms viral spikes on the S domain and contains determinants for antigenicity and host-cell interactions. The P domain binds carbohydrate structures, i.e., histo-blood group antigens, which are thought to be important for norovirus infections. In this protocol, we describe a method for producing high quality norovirus P domains in high yields. These proteins can then be used for X-ray crystallography and ELISA in order to study antigenicity and host-cell interactions.
The P domain is firstly cloned into an expression vector and then expressed in bacteria. The protein is purified using three steps that involve immobilized metal-ion affinity chromatography and size exclusion chromatography. In principle, it is possible to clone, express, purify, and crystallize proteins in less than four weeks, which makes this protocol a rapid system for analyzing newly emerging norovirus strains.

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

Here, we describe a method to express and purify high quality norovirus protruding (P) domains in E. coli for use in X-ray crystallography studies. This method can be applied to other calicivirus P domains, as well as non-structural proteins, i.e., viral protein genome-linked (VPg), protease, and RNA dependent RNA polymerase (RdRp).

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. ...

High-throughput Measurement of Gut Transit Time Using Larval Zebrafish

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and pharmacological similarities to that of the mammals. GI intolerance during clinical testing of drug candidates is common and may pose a serious threat to human health. Testing for GI toxicity in preclinical mammalian models can be expensive in terms of time, test compound, and labor. The high-throughput method presented here may be used to predict GI safety issues. Compared to mammalian models, this method allows for more expedient assessment of test compound effects on GI transit while using low quantities of compound. In this method, larval zebrafish (7 days post fertilization) are fed food containing a fluorescent label. After feeding, each larval fish is placed into a well of a 96-conical-bottom-well plate and dosed with test compound (dissolved in water) or the vehicle. As gut transit occurs, fecal matter accumulates on the bottom of the wells, and the rate at which this happens is monitored by measuring fluorescence from the bottom of the well repeatedly over time using a plate spectrophotometer. The fluorescence from larvae in a given treatment group are averaged and these values are graphed along with standard error, for each measurement time, yielding a curve representing average transit of food over time. Effects on gut transit time are identified by comparing the area under the curve for each treatment group to that of the vehicle-treated group. This method detected changes in zebrafish GI transit time induced by drugs with known clinical GI effects; it can be employed to interrogate dozens of treatments for GI effects per day. As such, safer compounds can be quickly prioritized for mammalian testing, which expedites discovery and proffers 3Rs advancement.

The goal of this protocol is to measure the transit time of fluorescently labeled food through the gut of larval zebrafish in a high throughput fashion.

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and ...

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

Dosimetry for Cell Irradiation using Orthovoltage (40-300 kV) X-Ray Facilities

The importance of dosimetry protocols and standards for radiobiological studies is self-evident. Several protocols have been proposed for dose determination using low energy X-ray facilities, but depending on the irradiation configurations, samples, materials or beam quality, it is sometimes difficult to know which protocol is the most appropriate to employ. We, therefore, propose a dosimetry protocol for cell irradiations using low energy X-ray facility. The aim of this method is to perform the dose estimation at the level of the cell monolayer to make it as close as possible to real cell irradiation conditions. The different steps of the protocol are as follows: determination of the irradiation parameters (high voltage, intensity, cell container etc.), determination of the beam quality index (high voltage-half value layer couple), dose rate measurement with ionization chamber calibrated in air kerma conditions, quantification of the attenuation and scattering of the cell culture medium with EBT3 radiochromic films, and determination of the dose rate at the cellular level. This methodology must be performed for each new cell irradiation configuration as the modification of only one parameter can strongly impact the real dose deposition at the level of the cell monolayer, particularly involving low energy X-rays.

Dosimetry for Cell Irradiation using Orthovoltage 40-300 kV X-Ray Facilities

This document describes a new dosimetry protocol for cell irradiations using low energy X-ray equipment. Measurements are performed in conditions simulating real cell irradiation conditions as much as possible.

The importance of dosimetry protocols and standards for radiobiological studies is self-evident. Several protocols have been proposed for dose ...

A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these techniques, cryo soft X-ray tomography (SXT) allows imaging whole cryo-preserved cells in the water window X-ray energy range (284-543 eV), in which carbon structures have intrinsically higher absorption than water, allowing the 3D reconstruction of the linear absorption coefficient of the material contained in each voxel. Quantitative structural information at the level of whole cells up to 10 &#181;m thick is then achievable this way, with high throughput and spatial resolution down to 25-30 nm half-pitch. Cryo-SXT has proven itself relevant to current biomedical research, providing 3D information on cellular infection processes (virus, bacteria, or parasites), morphological changes due to diseases (such as recessive genetic diseases) and helping us understand drug action at the cellular level, or locating specific structures in the 3D cellular environment. In addition, by taking advantage of the tunable wavelength at synchrotron facilities, spectro-microscopy or its 3D counterpart, spectro-tomography, can also be used to image and quantify specific elements in the cell, such as calcium in biomineralization processes. Cryo-SXT provides complementary information to other biological imaging techniques such as electron microscopy, X-ray fluorescence or visible light fluorescence, and is generally used as a partner method for 2D or 3D correlative imaging at cryogenic conditions in order to link function, location, and morphology.

Here, a protocol describing the sample preparation and data collection steps required in cryo soft X-ray tomography (SXT) to image the ultrastructure of whole cryo-preserved cells at a resolution of 25 nm half pitch, is presented.

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these ...