Name: rapid acquisition of 3d images using high-resolution episcopic microscopy
Uploaded: 2016-11-21T13:00:00
Description: Watch this Scientific Journal Video about Rapid Acquisition of 3D Images Using High-resolution Episcopic Microscopy at JoVE.com

We thank Drs. Yichen Huang, Chunming Guo and Zhenfang Zhou for their technical support. This research was funded by NIH/NIDDK (1R01DK091645-01A1, XL) and American Heart Association (AHA, 13GRNT16950006, XL).

All animal uses are approved by the Institutional Animal Care and Use Committee at Boston Children&#39;s Hospital.
1. Sample Preparation
<ol>
	<li>Timed mating
	<ol>
		<li>Place a C57Bl6 wild type male and a female together in the same cage.</li>
		<li>Check for the formation of a mating plug early next morning. The mating plug (aka, vaginal or copulation plug) is a hardened gelatinous deposition that blocks the vagina.</li>
		<li>Set the plug date as embryonic day ...

<ol>
	<li>Gregg, C. L., Butcher, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+in+vivo+imaging+of+embryonic+development:+opportunities+and+challenges.">Quantitative in vivo imaging of embryonic development: opportunities and challenges.</a> Differentiation. 84, 149-162 (2012).</li><li>Liu, X., Tobita, K., Francis, R. J., Lo, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+techniques+for+visualizing+and+phenotyping+congenital+heart+defects+in+murine+models.">Imaging techniques for visualizing and phenotyping congenital heart defects in murine models.</a> Birth defects Res C. 99, 93-105 (2013).</li><li>Norris, F. 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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=Episcopic+three-dimensional+imaging+of+embryos.">Episcopic three-dimensional imaging of embryos.</a> Cold Spring Harb protoc. , 641-646 (2012).</li><li>Sizarov, A., 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=Three-dimensional+and+molecular+analysis+of+the+arterial+pole+of+the+developing+human+heart.">Three-dimensional and molecular analysis of the arterial pole of the developing human heart.</a> J Anat. 220, 336-349 (2012).</li><li>Anderson, R. 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Here, we present a modified protocol using routine laboratory equipment to acquire serial HREM images that are compatible for rapid 3D visualization and morphometric analysis of complex structures. Because the high-resolution images are taken directly from the block face instead of individual sections, the fine morphological features are preserved and rapidly reconstructed digitally in a 3D visualization program.
3D imaging offers significant advantages in visualizing and understanding complex...

The advent of 3D imaging technologies has opened the possibility of systemic analysis of detailed morphological features during fetal development. A variety of 3D imaging modalities is now available include micro-magnetic resonance imaging (micro-MRI), micro computed tomography, high frequency ultrasound, optical coherence tomography, optical project tomography and episcopic microscopy1-4. Each modality has its own unique features in resolution, contrast, speed, cost, in utero capability and availability.
Episcopic fluorescence image capture (EFIC) and high-resolution episcopic microscopy (HREM) are episcopic microscopy imaging methods4. Here, high-resolution serial images are captured continuously from the block face instead of tissue sections. The resulting images faithfully reflect detailed morphological features with minimal or no tissue distortion. The acquired volumetric data is readily converted to high-resolution 3-D images suitable for accurate morphometric analysis. Because of relative low cost and high resolution, HREM and EPIC have become the superior alternatives for systemic assessment of mouse models of human birth defects2,4-8.
Both EFIC and HREM methods require specimens to be embedded in the suitable medium. EFIC detects autofluorescence emitted from the embedded tissue. Because of this, it is limited to specimens emitting high levels of autofluorescence but not those with relatively weak autofluorescence such as early stage embryos9. To overcome the dependency of autofluorescence, specimens are stained with a fluorescent dye such as eosin for HREM imaging. The choices of dyes and staining methods also make HREM more compatible to detect molecular signals in the context of tissue architecture and morphology4,10.
In this article, we present an improved HREM protocol suitable for whole body assessment of the late stage mouse embryos. To facilitate staining, we include a pretreatment step to increase tissue penetration, thereby enabling HREM imaging of older mouse embryos.

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			<td height="21" style="height:21px;width:377px;">Mice</td>
			<td style="width:356px;">The Jackson Laboratory</td>
			<td style="width:136px;">C57BL6</td>
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			<td height="21" style="height:21px;width:377px;">Ethyl Alcohol</td>
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			<td height="21" style="height:21px;">Urea</td>
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			<td height="21" style="height:21px;">JB-4 Embedding Kit - Solution A</td>
			<td>Polysciences, Inc.</td>
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			<td>Polysciences, Inc.</td>
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			<td height="21" style="height:21px;">JB-4&#160;Embedding Kit - Catalyst</td>
			<td>Polysciences, Inc.</td>
			<td style="width:136px;">02618-12</td>
			<td>Infiltration Solution</td>
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			<td height="21" style="height:21px;">Embedding Molds</td>
			<td>Polysciences, Inc.</td>
			<td style="width:136px;">23185-1</td>
			<td>16x8mm</td>
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			<td height="21" style="height:21px;">Peel-A-Way Sharp Embedding Molds</td>
			<td>Polysciences, Inc.</td>
			<td style="width:136px;">18986-1</td>
			<td>22mm x 22mm square, truncated to 12mm x 12mm</td>
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			<td>Polysciences, Inc.</td>
			<td style="width:136px;">15899</td>
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			<td>Embryo dissection</td>
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			<td style="width:356px;">&#160;ROBOZ</td>
			<td style="width:136px;">RS-5153</td>
			<td>Embryo dissection</td>
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			<td height="21" style="height:21px;">Delicate Operating Scissor</td>
			<td style="width:356px;">&#160;ROBOZ</td>
			<td style="width:136px;">RS-6700</td>
			<td>Embryo dissection</td>
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		<tr height="21">
			<td height="21" style="height:21px;">14 ml Polypropylene Round-Bottom Tubes</td>
			<td style="width:356px;">Falcon</td>
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			<td height="21" style="height:21px;">50 ml Polypropylene Conical Tubes</td>
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			<td style="width:356px;">MP Biomedicals</td>
			<td style="width:136px;">102892</td>
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			<td>PBS Solution</td>
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			<td>Sigma</td>
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			<td>PBS Solution</td>
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			<td height="21" style="height:21px;width:377px;">Sodium phosphate dibasic heptahydrate</td>
			<td style="width:356px;">Sigma-Aldrich</td>
			<td style="width:136px;">S9390</td>
			<td>PBS Solution</td>
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			<td height="21" style="height:21px;width:377px;">Digital Camera</td>
			<td style="width:356px;">Hamamatsu Photonics</td>
			<td style="width:136px;">C11440-10C</td>
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			<td height="21" style="height:21px;width:377px;">Microtome</td>
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			<td>Carl Zeiss</td>
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			<td></td>
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			<td>Olympus</td>
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			<td height="21" style="height:21px;width:377px;">Fiber Optic Light Source</td>
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			<td style="width:136px;">KL 1500 LCD</td>
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			<td style="width:136px;">95057-832</td>
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			<td style="width:136px;">pro 2012</td>
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			<td height="21" style="height:21px;width:377px;">Photoshop</td>
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			<td height="21" style="height:21px;width:377px;">Amira 3D Software</td>
			<td style="width:356px;">Visage Imaging</td>
			<td style="width:136px;">5.4</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:377px;">Computer</td>
			<td style="width:356px;">Dell T7600, 2x900GB SAS hard drive and 64 GB DDR3 RDIMM memory</td>
			<td style="width:136px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:377px;">Rocking Platform Shakers</td>
			<td style="width:356px;">VWR</td>
			<td style="width:136px;">40000-304</td>
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			<td height="21" style="height:21px;width:377px;">Standard Hot Plate Stirrer</td>
			<td style="width:356px;">VWR</td>
			<td style="width:136px;">12365-382</td>
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			<td height="21" style="height:21px;width:377px;">Analytical Balance</td>
			<td style="width:356px;">Mettler Toledo</td>
			<td style="width:136px;">AB54-S</td>
			<td></td>
		</tr>
	</tbody>
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rapid acquisition of 3d images using high-resolution episcopic microscopy

High-resolution episcopic microscopic (HREM) technology enables rapid acquisition of high-resolution digital volumetric and three-dimensional (3D) morphometric data. Here, we describe the detailed protocol to image the entire mouse embryo. The protocol consists of four major sections: sample preparation, embedding, image acquisition and finally, 3D visualization. The technology requires specimens to be stained with a fluorescent dye, which can be problematic for large or dense specimens. To overcome this limitation, we have improved the existing protocol to enhance tissue penetration of the dye by pretreating the specimen with a solution containing urea and sodium dodecyl sulfate. The protocol uses only routine laboratory equipment and reagents for easy adaptation in standard laboratory settings. We show that the resulting high-resolution 3D images faithfully recapitulate the detailed morphologic features of the internal organs of mouse embryos, thereby permitting morphometric analyses. Together, we present a detailed and improved protocol using standard laboratory equipment to acquire high-resolution 3D images of small and large sized specimens.

We have reported high quality HREM images of mouse embryos between e9.5 and e13.58. Image quality of the late staged embryos, however, is significantly compromised because of the limited tissue penetration of the fluorescent dye eosin. To increase the staining efficiency, we have tested several pretreatment methods that are compatible to fluorescent imaging11. Specifically, the e15.5 mouse embryos were treated with either Sca/e A212 or Whole-Body CUBIC<sup...

Watch this Scientific Journal Video about Rapid Acquisition of 3D Images Using High-resolution Episcopic Microscopy at JoVE.com

Rapid Acquisition of 3D Images Using High-resolution Episcopic Microscopy

We describe a detailed protocol using high-resolution episcopic microscopy to acquire three-dimensional (3D) images of mouse embryos. This improved protocol utilizes a modified tissue preparation method to enhance penetration of the fluorescent dye, thereby permitting morphometric analysis of both small and large-sized specimens.

High-resolution episcopic microscopic (HREM) technology enables rapid acquisition of high-resolution digital volumetric and three-dimensional (3D) ...

The overall goal of this protocol is to rapidly acquire high-resolution 3D digital images of biological specimens using standard laboratory equipment. This method can answer key questions in the field of developmental biology through the visualization and dynamics of complex morphological features. The main advantages of this technique are that it uses standard lab equipment and requires minimum prior experience.Demonstrating the procedure will be Jungang Huang, a graduate student from my lab. After dissection and fixation of the embryo as described in the text protocol, replace the fixative with clearing solution, and gently rotate the specimen on a rocker at room temperature for one to two weeks. At the end of the incubation, transfer the specimen to 10%formalin and leave on the rocker for another two days.On the third day, wash the sample in PBS for three five-minute washes. Then dehydrate the samples in an ascending ethanol series at room temperature, on a gentle rocker for two hours for each concentration. After the 100%ethanol dehydration, transfer the specimen to staining solution for one hour, protected from light.Then, replace the solution with fresh staining solution for overnight labeling of the specimen. The next morning, immerse the sample in a one to one volume to volume mixture of freshly prepared infiltration and staining solutions for at least three hours on a rocker at four degrees Celsius. At the end of the incubation, transfer the embryo into infiltration solution for another three-hour rocking incubation.Then replace the supernatant with fresh infiltration solution for three more days of rocking at four degrees Celsius. On the fourth day, orient the embryo in an embedding mold and place the mold on ice. Mix solution B with the infiltration solution and then gently submerge the mold in cold embedding solution at four degrees Celsius.After about three hours, use a tip to confirm that the solution has solidified, and push the specimen out of the mold. Trim any excess embedding material, then reorient the specimen block in a new mold in a cross orientation. Next, place the entire setup on ice in a fume hood.Gently add embedding solution to the new mold until the mold is submerged. Then, place a block holder on top of the mold. After three to four hours, peel away the embedding mold and place the block under a stereo microscope with oblique illumination to determine the position of the specimen within the block.On the block face, mark grid lines around the specimen and use the lines as guides for trimming any excess embedding material. To image the specimen, first clamp the block to a microtome, and obtain a fresh surface cut of the specimen. Then mount a stereo zoom microscope horizontally facing the block face of the specimen, and turn on the microscope and the accompanying computer.Next, start the image acquisition software and use the live view mode in imaging software to focus the optics to the block face. Align the microscope and microtome as needed, so that the block face is at the center of viewfinder. One of the most critical steps of this procedure is to acquire images at the fixed home position of the microtome.Adjust the zoom to make sure the entire block face is within the viewfinder. Then select the green fluorescent protein filter. Proceed to pre-cut the block until the specimen tissue appears, and re-focus the image as needed.After measuring and setting the exposure time manually, acquire and save images of the block face after each fresh cut, and record the important image acquisition parameters. At the end of the imaging session, export and convert all of the image files to JPEG format, and use the appropriate image processing software to invert all of the original images, adjusting the brightness and contrast of the images as necessary. Upload all the processed images to a 3D visualization software, following the instructions.Align all images using the automatic mode, and show the 3D and 2D results. In this representative image of a whole-mount surface view of an embryonic day 15.5 mouse embryo prepared and imaged as just demonstrated. The detailed morphological features of the skin, including the whisker pads and developing hair follicles can be observed.Importantly, the resolution of the virtual section view of an embryonic day 15.5 specimen imaged by this method is similar to a standard histological section. Once master the entire procedure after the pretreatment with the clearing solution can be completed in one week, if it is performed properly. While attempting this procedure, it is important to remember to keep the staining solution protect from light at four degree during infiltration and embedding step.Following this procedure, other methods, like scale staining, can be performed to answer additional question about cell types'specific distribution patterns. After watching this video, you should have a good understanding of how to use laboratory equipment to acquire high-resolution 3D images of biological specimens for their morphometric analysis. Don't forget that working with fixation, staining, and infiltration solution can be extremely hazardous, and that precaution such as operating in a fume hood and wearing gloves should always be taken while performing this procedure.

Research

JoVE Journal

Developmental Biology

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

High-resolution Episcopic Microscopy (HREM) - Simple and Robust Protocols for Processing and Visualizing Organic Materials

High-resolution Episcopic Microscopy HREM - Simple and Robust Protocols for Processing and Visualizing Organic Materials

We provide simple protocols for generating digital volume data with the high-resolution episcopic microscopy (HREM) method. HREM is capable of imaging ...

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

Visualization and Analysis of Pharyngeal Arch Arteries using Whole-mount Immunohistochemistry and 3D Reconstruction

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart ...

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans ...