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>Polysciences, Inc.</td>
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			<td>Polysciences, Inc.</td>
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			<td>Embryo dissection</td>
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			<td style="width:136px;">RS-6700</td>
			<td>Embryo dissection</td>
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			<td height="21" style="height:21px;">14 ml Polypropylene Round-Bottom Tubes</td>
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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) ...

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