The authors would like to thank the Agriculture, Fisheries and Conservation Department of the Hong Kong Special Administrative Region Government for the continuous support in this project. Sincere appreciation is also extended to veterinarians, staff, and volunteers from the Aquatic Animal Virtopsy Lab, City University of Hong Kong, Ocean Park Conservation Foundation Hong Kong and Ocean Park Hong Kong&#160;for paying great effort on the stranding response in this project. Special gratitude is owed to technicians in CityU Veterinary Medical Centre and Hong Kong Veterinary Imaging Centre for operating the CT and MRI units for the present study. Any opinions, findings, conclusions or recommendations expressed herein do not necessarily reflect the views of the Marine Ecology Enhancement Fund or the Trustee. This project was funded by the Hong Kong Research Grants Council (Grant number: UGC/FDS17/M07/14), and the Marine Ecology Enhancement Fund (grant number: MEEF2017014, MEEF2017014A,&#160;MEEF2019010 and MEEF2019010A), Marine Ecology Enhancement Fund, Marine Ecology &#38; Fisheries Enhancement Funds Trustee Limited. Special thanks to Dr. Mar&#237;a Jos&#233; Robles Malagamba for English editing of this manuscript.

NOTE: In the framework of the Hong Kong cetacean virtopsy stranding response program, stranded cetaceans were routinely examined by PMCT. The authors were in charge of virtopsy scanning, data postprocessing (e.g., image reconstruction and rendering), data interpretation, and virtopsy reporting1. This advanced technology emphasizes attentive findings and gives insights on the initial investigation of PM findings prior to conventional necropsy (https://www.facebook.com/aquanimallab).
1. Data preparation
<ol>
	<li>Export the acquired CT datasets in DICOM 3.0 format. Copy the DICOM folder to computer (e.g., desktop).</li>
	<li>Open a free or commercial DICOM viewer. The following steps are based on the TeraRecon Aquarius iNtuition Workstation (version 4.4.12).</li>
	<li>Double-click the icon of Aquarius iNtuition Client Viewer (AQi) icon. Enter user name, password, and server name in the appropriate fields. Click the Login button. 
	NOTE: Make sure that the server name field has the correct server IP address.</li>
	<li>Click Import under the data management tool buttons and select the DICOM folder to import. Click the Update icon to renew the study list after the import status reaches 100%.</li>
	<li>View the datasets by selecting 1 or multiple CT series from the Patient List by double-left-clicking the series.</li>
	<li>After loading the designated series, click the Window Layout Button for the 2x2 display interface, showing a 2x2 default layout, a 3D volume rendered image (upper-right panel) and 3 MPR images in axial view (upper-left panel), coronal view (lower-left panel), sagittal view (lower-right panel), giving different orientations.</li>
	<li>Evaluate the virtopsy datasets thoroughly using different image rendering techniques provided.</li>
</ol>
2. Multiplanar reconstruction (MPR)
<ol>
	<li>Display the default MPR from axial view (upper-left panel), coronal view (lower-left panel), and sagittal view (lower-right panel) after loading the series. Change the rendering mode to MPR by either right-clicking the image and select MPR or click MPR in the Rendering Mode Mini-Toolbar.</li>
	<li>Evaluate the virtopsy datasets from the first image to the last image using the axial view, followed by coronal and sagittal views, with the assistance of the following functions: Click Slice, left-click-hold mouse button and drag the mouse to view and adjust the CT image slice by slice.</li>
	<li>Click Pan, left-click-hold mouse button and drag the mouse to adjust the location of the image inside the panel.</li>
	<li>Click Zoom, left-click-hold mouse button and drag the mouse to magnify or minify the image.</li>
	<li>Select the appropriate pre-set window/levels by clicking Abd 1 (window width: 350, window level: 75), Abd 2 (window width: 250, window level: 40), Head (window width: 100, window level: 45), Lung (window width: 1500, window level: -700), Bone (window width: 2200, window level: 200) in the Window/Level Mini-Toolbar, depending on the regions of interest.</li>
	<li>Click Window/Level (W/L), left-click-hold mouse button and drag the mouse to manually adjust the window width and window level of the CT slice.</li>
	<li>Click Rotate, left-click-hold mouse button and drag the mouse to rotate the MPR images.</li>
	<li>Left-click-hold mouse button on the center of MPR Crosshairs to concurrently adjust the regions of interest and slices in 3 MPR images. 
	NOTE: There are mouse modes for the 4 main functions of rotations, panning, zooming and window/level changes provided by AQi to facilitate the viewing process. For keyboard shortcuts, see Table 1.</li>
</ol>
3. Curved planar reformation (CPR)
<ol>
	<li>Decide the region of anatomical interest. Left-click-hold mouse button on the center of MPR crosshairs to the particular region of interest.</li>
	<li>View the MPR from 3 different views. Ensure the MPR crosshairs is placed in a correct location. Adjust the MPR crosshairs if it is not.</li>
	<li>Select 1 display panel from axial, coronal, and sagittal views as study panel, e.g., aiming to view the flipper from an axial view.</li>
	<li>Depending on the study panel, adjust the extended line of MPR crosshairs (e.g., blue color) from coronal view perpendicularly to the region of interest by left-click-hold mouse button on the rotation point of extended line.</li>
	<li>Adjust another extended line (e.g., red color) of MPR crosshairs from sagittal view parallel to the region of interest by left-click-hold mouse button on the rotation point of extended line.</li>
	<li>Look at the axial view to check whether the region of interest is adjusted correctly. Adjust the extended lines if it is not. Evaluate the virtopsy datasets using the 4 main functions of rotation, panning, zooming and window/level changes. 
	NOTE: There are 3 colored extended lines of MPR crosshairs (green, red, and blue), representing different alignments of the MPR plane (Figure 2).</li>
</ol>
4. Maximum intensity projection (MIP)
<ol>
	<li>Change the rendering mode to MIP by either right-clicking the image and selecting MIP or by clicking MIP in the Rendering Mode Mini-Toolbar.</li>
	<li>Adjust the slab thickness on the right upper corner (minimum: 1 mm, maximum: 500 mm) by clicking the green annotation and select a new thickness to visualize the regions of interest, e.g., the bronchial tree in the lung.</li>
	<li>Evaluate the virtopsy datasets using the 4 main functions of rotation, panning, zooming, and window/level changes.</li>
</ol>
5. Minimum intensity projection (MinIP)
<ol>
	<li>Change the rendering mode to MIP by either right-clicking the image and selecting MinIP or by clicking MinIP in the Rendering Mode Mini-Toolbar.</li>
	<li>Adjust the slab thickness on the right upper corner (minimum: 1 mm, maximum: 500 mm) by clicking the green annotation and select a new thickness to visualize the regions of interest (e.g., the bronchial tree in the lung).</li>
	<li>Evaluate the virtopsy datasets using the 4 main functions of rotation, panning, zooming, and window/level changes.</li>
</ol>
6. Direct volume rendering (DVR)
NOTE: As 1 of the default display 2x2 interfaces, DVR (upper-right panel) shows the 3D rendered images of the carcass. The default DVR template setting is AAA (abdominal aortic aneurysm; window width: 530, window level: 385), giving a gross skeletal structure of the carcass.
<ol>
	<li>Automatically adjust the windowing setting by clicking Template under the Viewer and select the appropriate DVR template, e.g., Gray 10% (window width: 442, window level: 115), Fracture (window width: 2228, window level: 1414) if needed.</li>
	<li>Click Window/Level (W/L), left-click-hold mouse button and drag the mouse to adjust the window width and window level of the CT slice manually, giving an outer layer (e.g., epidermal surface) to inner layer (e.g., internal structure).</li>
	<li>Use the 4 main functions of rotation, panning, zooming, and window/level changes for further corrections. 
	NOTE: All DVR templates provided by AQi are human clinical oriented, not designated for PM imaging of cetaceans.</li>
</ol>
7. Segmentation and Region-of-Interest (ROI) Editing
<ol>
	<li>Segment the CT image slice using 3 different tools, Slab and Cube View tool, Free ROI tool, and Dynamic region growing tool.</li>
	<li>For Slab and Cube View tool, click Slab under Tool, giving a parallel display line. Adjust the slab location by relocating the MPR crosshairs from the corresponding MPR views. Change the slab thickness (minimum: 1 mm, maximum: 500 mm) via the slab thickness bar, resulting a segmentation of 3D rendered images of the carcass.</li>
	<li>For Free ROI tool, click FreeRO under Tool. Hold on the Shift key on the keyboard, and use either Draw Free Curve on MPR, Draw Circle on MPR, or Draw Sphere on MPR to exclude/include the region of interest from the MPR views and DVR.</li>
	<li>For Dynamic region growing tool, click Region under Tool. Hold on the Shift key on keyboard, left-click-hold mouse button and scroll the middle button of the mouse (scroll-up: increase the selecting region, scroll-down: decrease the selecting region), giving a highlighted region. Click Exclude to delete the region. Click Include to keep the region.</li>
</ol>
8. Transfer Functions (TF)
<ol>
	<li>Click 3D Setting under Viewer, select Copy to create a new 3D reconstructed model.</li>
	<li>In the new 3D reconstructed model, click FreeRO or Region under Tool. Hold on the Shift key on the keyboard, use 3D VR to include the region of interest and then click Select.</li>
	<li>Configure the 3D settings, including W/L Slider, W/L Text-input Boxes, VR Pull-down Menu, Opacity Slider (minimum: 0, maximum: 1), Opacity Text-input Box, and HU Range Color Slider under 3D Setting.</li>
	<li>Right-click 1 of the sliders in the color slider bar to change the color of the DVR. Select Change Color and define a custom color from the color palette if needed.</li>
</ol>
9. Perspective Volume Rendering (PVR)
<ol>
	<li>To launch the Flythrough Module, right-click on the selected series and select Flythrough from the right-click menu.</li>
	<li>Choose the Primary 3D of Reading Style Preference Wizard for primary view selection. Click the 2x2 screen layout and OK, resulting in an automatically RVR, e.g., colon. Make sure the region of interest is selected.</li>
	<li>Build a flight path by placing the start and end of control points by drawing a path. Correct the path by clicking the Edit Connection/Edit Path radio button in the tool panel if there is a broken path or missing structure, editing the control points for smoother sections of the curve or correcting problems. Create new control points by clicking on the flight path. Once the flight path is correct, click OK.</li>
	<li>View the Flythrough window displayed, showing a Main flythrough window, MPR views and Flat view.</li>
	<li>Use Cine Tools by clicking the Tool Panel located on the right side of the screen to evaluate the luminal structure. Adjust the speed and direction of the flythrough using Fly backward, Pause, Fly Forward, Slow down flythrough, and Speed up flythrough under the Cine tools.</li>
</ol>
10. Data evaluation
<ol>
	<li>Conduct virtopsy evaluation systematically from head to tail. It is generally within 30 minutes, acting as a reference to guide veterinarians for subsequent necropsy.</li>
	<li>After necropsy, compare virtopsy findings and necropsy findings. Based on the site report, virtopsy, necropsy, and sample analysis (e.g., histopathology and microbiology), conclude the PM investigation on the biological health and profile of the stranded cetacean.</li>
</ol>

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<li>Errickson, D., Thompson, T. J. U., Rankin, B. W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+application+of+3D+visualization+of+osteological+trauma+for+the+courtroom%3A+a+critical+review%5BTitle%5D)+AND+%22Journal+of+Forensic+Radiology+and+Imaging%22%5BJournal%5D)">The application of 3D visualization of osteological trauma for the courtroom: a critical review.</a> Journal of Forensic Radiology and Imaging. 2 (3), 132-137 (2014).</li>
<li>Prokop, M., Galanski, M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aProkop%2C+M+%22Spiral+and+multislice+computed+tomography+of+the+body%22">Spiral and multislice computed tomography of the body</a>. , Thieme Medical Publishers. (2003).</li>
<li>Kawel, N., Seifert, B., Luetolf, M., Boehm, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+slab+thickness+on+the+CT+detection+of+pulmonary+nodules%3A+use+of+sliding+thin-slab+maximum+intensity+projection+and+volume+rendering%5BTitle%5D)+AND+%22American+Journal+of+Roentgenology%22%5BJournal%5D)">Effect of slab thickness on the CT detection of pulmonary nodules: use of sliding thin-slab maximum intensity projection and volume rendering.</a> American Journal of Roentgenology. 192 (5), 1324-1329 (2009).</li>
<li>Vlassenbroek, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+isotropic+imaging+and+computed+tomography+reconstructions%5BTitle%5D)+AND+%22Comparative+Interpretation+of+CT+and+Standard+Radiography+of+the+Chest%2C+Medical+Radiology%22%5BJournal%5D)">The use of isotropic imaging and computed tomography reconstructions.</a> Comparative Interpretation of CT and Standard Radiography of the Chest, Medical Radiology. , Springer-Verlag. Berlin Heidelberg. 53-73 (2011).</li>
<li>van Ooijen, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Noninvasive+coronary+imaging+using+electron+beam+CT%3A+surface+rendering+versus+volume+rendering%5BTitle%5D)+AND+%22American+Journal+of+Roentgenology%22%5BJournal%5D)">Noninvasive coronary imaging using electron beam CT: surface rendering versus volume rendering.</a> American Journal of Roentgenology. 180 (1), 223-226 (2003).</li>
<li>Remy-Jardin, M., Remy, J., Artaud, D., Fribourg, M., Duhamel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Volume+rendering+of+the+tracheobronchial+tree%3A+clinical+evaluation+of+bronchographic+images%5BTitle%5D)+AND+%22Radiology%22%5BJournal%5D)">Volume rendering of the tracheobronchial tree: clinical evaluation of bronchographic images.</a> Radiology. 208 (3), 761-770 (1998).</li>
<li>Bassett, J. T., Liotta, R. A., Barlow, D., Lee, D., Jensen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Colonic+perforation+during+screening+CT+colonography+using+automated+CO2+insufflation+in+an+asymptomatic+adult%5BTitle%5D)+AND+%22Abdominal+Imaging%22%5BJournal%5D)">Colonic perforation during screening CT colonography using automated CO2 insufflation in an asymptomatic adult.</a> Abdominal Imaging. 33 (5), 598-600 (2008).</li>
</ol>

The authors have nothing to disclose.

For the clear visualization of virtopsy datasets, 8 image rendering techniques, consisting of both 2D and 3D rendering, were routinely applied to each stranded carcass for the PM investigation of their biological health and profile. These rendering techniques included MPR, CPR, MIP, MinIP, DVR, segmentation, TF, and PVR. Diverse rendering techniques are complementarily used together with windowing adjustment. The concepts of each image reformation technique and advantages are also described.
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Virtopsy, also known as postmortem (PM) imaging, is the examination of a carcass with advanced cross-sectional imaging modalities, including postmortem computed tomography (PMCT), postmortem magnetic resonance imaging (PMMRI), and ultrasonography1. In humans, PMCT is useful in investigating traumatic cases of skeletal alterations2,3, foreign bodies, gaseous findings4,5,6, and pathologies of the vascular system7,8,9. Since 2014, virtopsy has been routinely implemented in the Hong Kong cetacean stranding response program1. PMCT and PMMRI are able to depict patho-morphological findings on carcasses that are too decomposed to be evaluated by conventional necropsy. The non-invasive radiological assessment is objective and digitally storable, allowing second opinion or retrospective studies years later1,10,11. Virtopsy has become a valuable alternative technique to provide new insights of PM findings in stranded marine animals12,13,14,15,16. Combined with necropsy, which is the gold standard to explain the pathophysiological reconstruction and cause of death17, the biological health and profile of the animals can be addressed. Virtopsy has been gradually recognized and implemented into stranding response programs worldwide, including but not limited to Costa Rica, Japan, Mainland China, New Zealand, Taiwan, Thailand and USA1.
Image rendering techniques in radiology use computer algorithms to transform numbers into information about the tissue. For example, radiological density is expressed in conventional X-rays and CT. The vast quantity of volumetric data is stored in the Digital Imaging and Communications in Medicine (DICOM) format. CT images can be used to produce isotropic voxel data using two-dimensional (2D) and three-dimensional (3D) image rendering in a postprocessing 3D workstation for high resolution visualization18,19. Quantitative data and results are mapped to transform serially acquired axial images into 3D images with gray-scale or color parameters19,20,21. Choosing an appropriate data visualization method from diverse rendering techniques is an essential technical determinant of the visualization quality, which significantly affects the analysis and interpretation of radiological findings21. This is particularly critical for stranding work that involves personnel without any radiology background, who need to understand the results in different circumstances17. The goal of implementing these image rendering techniques is to enhance the quality on the visualization of anatomical details, relationships and clinical findings, which boosts the diagnostic value of imaging and allows an effective rendition of the defined regions of interest17,19,22,23,24,25.
Although the primary axial CT/MRI images contain most information, they may limit accurate diagnosis or documentation of pathologies as structures cannot be viewed in various orthogonal planes. Image reformation at other anatomically aligned planes permits visualization of structural relationships from another perspective without having to reposition the body26. As medical anatomy and forensic pathology data are predominantly 3D in nature, color-coded PMCT images and 3D reconstructed images are preferred to gray-scale images and 2D slice images in view of improved understandability and suitability for courtroom adjudications27,28. With the advances in PMCT technology, a concern of visualization exploration (i.e., the creation and interpretation of 2D and 3D image) in cetacean PM investigation has been raised12,29. Various volumetric rendering techniques in the radiology workstation allow radiologists, technicians, referring clinicians (e.g., veterinarians and marine mammal scientists), and even laymen (e.g., stranding response personnel, government officers and general public) to visualize and study the regions of interest. Yet, the choice of a suitable technique and confusion of terminology remain a major issue. It is necessary to understand the basic concept, strengths and limitations of the common techniques, since it would significantly influence the diagnostic value and interpretation of radiological findings. Misuse of techniques may generate misleading images (e.g., images that have distortions, rendering errors, reconstruction noises or artefacts) and lead to an incorrect diagnosis30.
The present study aims to assess 8 essential image rendering techniques in PMCT that were used to identify most of the PM findings in stranded cetaceans in Hong Kong waters. Descriptions and practical examples of each technique are provided to guide radiologists, clinicians, and veterinarians worldwide through the often difficult and complicated realm of PMCT image rendering and review for the evaluation of biological health and profile.

<table><tbody><tr><td>Aquarius iNtuition workstation</td><td>TeraRecon Inc</td><td>NA</td><td></td></tr><tr><td>Siemens 64-row multi-slice spiral CT scanner Somatom go.Up</td><td>Siemens Healthineers</td><td>NA</td><td></td></tr></tbody></table>

image rendering techniques in postmortem computed tomography: evaluation of biological health and profile in stranded cetaceans

With 6 years of experience in implementing virtopsy routinely into the Hong Kong cetacean stranding response program, standardized virtopsy procedures, postmortem computed tomography (PMCT) acquisition, postprocessing, and evaluation were successfully established. In this pioneer cetacean virtopsy stranding response program, PMCT was performed on 193 stranded cetaceans, providing postmortem findings to aid necropsy and shed light on the biological health and profile of the animals. This study aimed to assess 8 image rendering techniques in PMCT, including multiplanar reconstruction, curved planar reformation, maximum intensity projection, minimum intensity projection, direct volume rendering, segmentation, transfer function, and perspective volume rendering. Illustrated with practical examples, these techniques were able to identify most of the PM findings in stranded cetaceans and served as a tool to investigate their biological health and profile. This study could guide radiologists, clinicians and veterinarians through the often difficult and complicated realm of PMCT image rendering and reviewing.

From January 2014 to May 2020, a total of 193 cetaceans that stranded in Hong Kong waters were examined by PMCT, including 42 Indo-Pacific humpback dolphins (Sousa chinensis), 130 Indo-Pacific finless porpoises (Neophocaena phocaenoides) and 21 other species. A whole-body scan was performed on 136 carcasses while 57 were partial scans on skulls and flippers. Anatomical features and pathologies commonly observed were illustrated with the 8 image rendering techniques for the evaluation of the stranded cet...

Watch this Scientific Journal Video about Image Rendering Techniques in Postmortem Computed Tomography: Evaluation of Biological Health and Profile in Stranded Cetaceans at JoVE.com

Image Rendering Techniques in Postmortem Computed Tomography: Evaluation of Biological Health and Profile in Stranded Cetaceans

The Hong Kong cetacean stranding response program has incorporated postmortem computed tomography, which provides valuable information on the biological health and profile of the deceased animals. This study describes 8 image rendering techniques that are essential for the identification and visualization of postmortem findings in stranded cetaceans, which will help clinicians, veterinarians and stranding response personnel worldwide to fully utilize the radiological modality.

With 6 years of experience in implementing virtopsy routinely into the Hong Kong cetacean stranding response program, standardized virtopsy procedures, ...

image-rendering-techniques-postmortem-computed-tomography-evaluation

Research

JoVE Journal

Biology

8.6K Views.  City University of Hong Kong. Deze studie heeft tot doel verschillende beeldweergavetechnieken in de postmortem computertomografie te beoordelen voor de evaluatie van de biologische gezondheid en het profiel in gestrande walvisachtigen in de wateren van Hongkong. Dit helpt radiologen, clinici en dierenartsen door het vaak moeilijke en ingewikkelde rijk van de postmortem computertomtom beeldretoriek en beoordeling te begeleiden. Acht beeldweergavetechnieken bestaan uit zowel 2D- als 3D-rendering die routinematig worden toe...

Video: Image Rendering Techniques in Postmortem Computed Tomography: Evaluation of Biological Health and Profile in Stranded Cetaceans

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

Bioengineering

Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for imaging vasculature, few are able to visualize the vascular network as a whole while extending to a resolution that includes the smaller vessels1,2. Additionally, many vascular casting techniques destroy the surrounding tissue, preventing further analysis of the sample3-5. One method which circumvents these issues is micro-Computed Tomography (&mu;CT). &mu;CT imaging can scan at resolutions &lt;10 microns, is capable of producing 3D reconstructions of the vascular network, and leaves the tissue intact for subsequent analysis (e.g., histology and morphometry)6-11. However, imaging vessels by ex vivo &mu;CT methods requires that the vessels be filled with a radiopaque compound. As such, the accurate representation of vasculature produced by &mu;CT imaging is contingent upon reliable and complete filling of the vessels. In this protocol, we describe a technique for filling mouse coronary vessels in preparation for &mu;CT imaging.
Two predominate techniques exist for filling the coronary vasculature: in vivo via cannulation and retrograde perfusion of the aorta (or a branch off the aortic arch) 12-14, or ex vivo via a Langendorff perfusion system 15-17. Here we describe an in vivo aortic cannulation method which has been specifically designed to ensure filling of all vessels. We use a low viscosity radiopaque compound called Microfil which can perfuse through the smallest vessels to fill all the capillaries, as well as both the arterial and venous sides of the vascular network. Vessels are perfused with buffer using a pressurized perfusion system, and then filled with Microfil. To ensure that Microfil fills the small higher resistance vessels, we ligate the large branches emanating from the aorta, which diverts the Microfil into the coronaries. Once filling is complete, to prevent the elastic nature of cardiac tissue from squeezing Microfil out of some vessels, we ligate accessible major vascular exit points immediately after filling. Therefore, our technique is optimized for complete filling and maximum retention of the filling agent, enabling visualization of the complete coronary vascular network &#x2013; arteries, capillaries, and veins alike.

Visualization of the coronary vessels is critical to advancing our understanding of cardiovascular diseases. Here we describe a method for perfusing murine coronary vasculature with a radiopaque silicone rubber (Microfil), in preparation for micro-Computed Tomography (&mu;CT) imaging.

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for ...

Medicine

Microfocus X-ray CT (microCT) Imaging of Actinia equina (Cnidaria), Harmothoe sp. (Annelida), and Xenoturbella japonica (Xenacoelomorpha)

Traditionally, biologists have had to rely on destructive methods such as sectioning in order to investigate the internal structures of opaque organisms. Non-destructive microfocus X-ray computed tomography (microCT) imaging has become a powerful and emerging protocol in biology, due to technological advancements in sample staining methods and innovations in microCT hardware, processing computers, and data analysis software. However, this protocol is not commonly used, as it is in the medical and industrial fields. One of the reasons for this limited use is the lack of a simple and comprehensible manual that covers all of the necessary steps: sample collection, fixation, staining, mounting, scanning, and data analyses. Another reason is the vast diversity of metazoans, particularly marine invertebrates. Because of marine invertebrates&#8217; diverse sizes, morphologies, and physiologies, it is crucial to adjust experimental conditions and hardware configurations at each step, depending on the sample. Here, microCT imaging methods are explained in detail using three phylogenetically diverse marine invertebrates: Actinia equina (Anthozoa, Cnidaria), Harmothoe sp. (Polychaeta, Annelida), and Xenoturbella japonica (Xenoturbellida, Xenacoelomorpha). Suggestions on performing microCT imaging on various animals are also provided.

Microfocus X-ray CT microCT Imaging of Actinia equina Cnidaria, Harmothoe sp. Annelida, and Xenoturbella japonica Xenacoelomorpha

Here, protocols for performing microfocus X-ray computed tomography (microCT) imaging of three marine invertebrate animals are explained in detail. This study describes steps such as sample fixation, staining, mounting, scanning, image reconstruction, and data analyses. Suggestions on how the protocol can be adjusted for different samples are also provided.

Traditionally, biologists have had to rely on destructive methods such as sectioning in order to investigate the internal structures of opaque organisms. ...

Environment

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro&#8211;computed tomography (&#181;CT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and &#181;CT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, &#181;CT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.
The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7th and 10th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 &#181;m) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

Here, we present a protocol to obtain high resolution micro computed tomography images of healthy and pathological large mammalian whole hearts with collagen-selective contrast enhancement.

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional ...

High-Resolution Cardiac Positron Emission Tomography/Computed Tomography for Small Animals

Positron emission tomography (PET) and computed tomography (CT) are among the most employed diagnostic imaging techniques, and both serve in understanding cardiac function and metabolism. In preclinical research, dedicated scanners with high sensitivity and high spatio-temporal resolution are employed, designed to cope with the demanding technological requirements posed by the small heart size and very high heart rates of mice and rats. In this paper, a bimodal cardiac PET/CT imaging protocol for experimental mouse and/or rat models of cardiac diseases is described, from animal preparation and image acquisition and reconstruction to image processing and visualization.
In particular, the 18F-labeled fluorodeoxyglucose ([18F]FDG)-PET scan allows for the measurement and visualization of glucose metabolism in the different segments of the left ventricle (LV). Polar maps are convenient tools to display this information. The CT part consists of a time-resolved 3D reconstruction of the entire heart (4D-CT) using retrospective gating without electrocardiography (ECG) leads, allowing the morphofunctional evaluation of the LV and the subsequent quantification of the most important cardiac function parameters, such as ejection fraction (EF) and stroke volume (SV). Using an integrated PET/CT scanner, this protocol can be executed within the same anesthesia induction without the need to reposition the animal between different scanners. Hence, PET/CT can be seen as a comprehensive tool for the morphofunctional and metabolic evaluation of the heart in several small animal models of cardiac diseases.

Here, we present an experimental imaging protocol for the quantification of cardiac function and morphology using high-resolution positron emission tomography/computed tomography for small animals. Both mice and rats are considered, discussing the different requirements of computed tomography contrast agents for the two species.

Positron emission tomography (PET) and computed tomography (CT) are among the most employed diagnostic imaging techniques, and both serve in understanding ...

Photogrammetric Three-dimensional Modeling and Printing of Cetacean Skeleton using an Omura's Whale Stranded in Hong Kong Waters as an Example

The preparation of cetacean, in particular baleen whale, skeletons presents a great challenge due to their high lipid content and uncommon size. Documentation of the skeletal morphology is important to produce accurate and reliable models for both research and educational purposes. In this paper, we used a 10.8-meter long Omura&#8217;s whale stranded in Hong Kong waters in 2014 as an example for the illustration. This rare and enormous specimen was defleshed, macerated, and sun-dried to yield the skeleton for research and public display. Morphology of each bone was then documented by photogrammetry. The complex contour of the skeleton made automated photoshoot inadequate and 3 manual methods were used on bones of different sizes and shapes. The captured photos were processed to generate three-dimensional (3D) models of 166 individual bones. The skeleton was printed half-size with polylactic acid for display purposes, which was easier to maintain&#160;than the actual cetacean bones with high residual fat content. The printed bones reflected most anatomical features of the specimen, including the bowing out rostral region and the caudal condylar facet that articulated with Ce1, yet the foramina on the parieto-squamosal suture, which are diagnostic character of Balaenoptera omurai, and an indented groove on the frontal bone at the posterior end of the lateral edge were not clearly presented. Extra photoshoots or 3D surface scanning should be performed on areas with meticulous details to improve precision of the models. The electronic files of the 3D skeleton were published online to reach a global audience and facilitate scientific collaboration among researchers worldwide.

The bone morphology of a semi-degreased baleen whale was documented by photogrammetry with a DSLR camera to generate three-dimensional (3D) models by computer, which were 3D printed as half-sized replicas of the original for display and educational purposes.

The preparation of cetacean, in particular baleen whale, skeletons presents a great challenge due to their high lipid content and uncommon size. ...

Longitudinal Quantification of Cardiovascular Calcification Using PET/CT Imaging

Novel Quantification Protocol for Cardiovascular Calcification Progression Using Longitudinal MicroPET/MicroCT Images

Micro positron emission tomography (PET) and micro computed tomography (CT) imaging are powerful, ideal research tools for following the progression of cardiovascular calcification. Due to their non-invasive nature, small research animals can be imaged at multiple time points. The challenge lies in the accurate quantification of cardiovascular calcification. Here, we provide a protocol, using images from the later disease stages as a template, to accurately quantify the progression of cardiovascular calcification in longitudinal studies. The protocol involves 1) the alignment of the chest area in multiple images from the same animal during a longitudinal study as the first step, 2) the definition of a region of interest (ROI) situated within the heart and the aorta at the site of larger calcium deposits that become apparent in later images, and 3) simultaneous segmentation and quantification of calcium deposits across all images acquired during the longitudinal study. This streamlined method enhances the accuracy of image analysis in following the progression of cardiovascular calcification by improving the precision of ROI definition and reducing the variability associated with earlier techniques that analyze individual scans independently.

Author Spotlight: Enhanced Quantification of Cardiovascular Calcification Progression for Longitudinal Micro PET/CT Studies in Small Research Animals

This novel protocol entails the quantification of cardiovascular calcification progression from serial micro positron emission tomography (PET)/micro computed tomography (CT) images in small research animals.

Micro positron emission tomography (PET) and micro computed tomography (CT) imaging are powerful, ideal research tools for following the progression of ...

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is a growing global health issue, and the impact of NAFLD is compounded by the current lack of effective treatments. Considerable limiting factors hindering the timely and accurate diagnosis (including grading) and monitoring of NAFLD, as well as the development of potential therapies, are the current inadequacies in the characterization of the hepatic microenvironment structure and the scoring of the disease stage in a spatiotemporal and non-invasive manner. Using a diet-induced NAFLD mouse model, we investigated the use of in vivo micro-computed tomography (CT) imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation. This imaging methodology allows for longitudinal analysis of liver steatosis and functional tissue uptake, as well as the evaluation of the relative blood volume, portal vein diameter, and density of the vascular network. Understanding the adaptations of the hepatic vascular network during NAFLD progression and correlating this with other ways of characterizing the disease progression (steatosis, inflammation, fibrosis) using the proposed method can pave the way toward the establishment of new, more efficient, and reproducible approaches for NAFLD research in mice. This protocol is also expected to upgrade the value of preclinical animal models for investigating&#160;the development of novel therapies against disease progression.

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

Using a diet-induced non-alcoholic fatty liver disease (NAFLD) mouse model, we describe the use of novel in vivo micro-computed tomography imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation.

Image Rendering Techniques in Postmortem Computed Tomography: Evaluation of Biological Health and Profile in Stranded Cetaceans

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