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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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+application+of+3D+visualization+of+osteological+trauma+for+the+courtroom:+a+critical+review.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Spiral and multislice computed tomography of the body. , (2003).</li><li>Kawel, N., Seifert, B., Luetolf, M., Boehm, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+slab+thickness+on+the+CT+detection+of+pulmonary+nodules:+use+of+sliding+thin-slab+maximum+intensity+projection+and+volume+rendering.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+isotropic+imaging+and+computed+tomography+reconstructions.">The use of isotropic imaging and computed tomography reconstructions.</a> Comparative Interpretation of CT and Standard Radiography of the Chest, Medical Radiology. , 53-73 (2011).</li><li>van Ooijen, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+coronary+imaging+using+electron+beam+CT:+surface+rendering+versus+volume+rendering.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume+rendering+of+the+tracheobronchial+tree:+clinical+evaluation+of+bronchographic+images.">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. 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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.
<...

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

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8.3K Views.  City University of Hong Kong. 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 moda...

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

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &amp;#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...