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.

rapid-acquisition-3d-images-using-high-resolution-episcopic

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. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

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

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

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 described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

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 compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

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

We provide simple protocols for generating digital volume data with the high-resolution episcopic microscopy (HREM) method. HREM is capable of imaging organic materials with volumes up to 5 x 5 x 7 mm3 in typical numeric resolutions between 1 x 1 x 1 and 5 x 5 x 5 &#181;m3. Specimens are embedded in methacrylate resin and sectioned on a microtome. After each section an image of the block surface is captured with a digital video camera that sits on the phototube connected to the compound microscope head. The optical axis passes through a green fluorescent protein (GFP) filter cube and is aligned with a position, at which the bock holder arm comes to rest after each section. In this way, a series of inherently aligned digital images, displaying subsequent block surfaces are produced. Loading such an image series in three-dimensional (3D) visualization software facilitates the immediate conversion to digital volume data, which permit virtual sectioning in various orthogonal and oblique planes and the creation of volume and surface rendered computer models. We present three simple, tissue specific protocols for processing various groups of organic specimens, including mouse, chick, quail, frog and zebra fish embryos, human biopsy material, uncoated paper and skin replacement material.

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

We provide simple and robust protocols for processing biopsy material of various species, embryos of biomedical model organisms and samples of other organic tissues in order to permit digital volume data generation with the high-resolution episcopic microscopy method.

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 combination of light and electron microscopy. Samples embedded in resin are sectioned into arrays consisting of ribbons of hundreds of ultrathin sections and deposited on pieces of silicon wafer or conductively coated coverslips. Arrays are imaged at low resolution using a digital consumer like smartphone camera or light microscope (LM) for a rapid large area overview, or a wide field fluorescence microscope (fluorescence light microscopy (FLM)) after labeling with fluorophores. After post-staining with heavy metals, arrays are imaged in a scanning electron microscope (SEM). Selection of targets is possible from 3D reconstructions generated by FLM or from 3D reconstructions made from the SEM image stacks at intermediate resolution if no fluorescent markers are available. For ultrastructural analysis, selected targets are finally recorded in the SEM at high-resolution (a few nanometer image pixels). A ribbon-handling tool that can be retrofitted to any ultramicrotome is demonstrated. It helps with array production and substrate removal from the sectioning knife boat. A software platform that allows automated imaging of arrays in the SEM is discussed. Compared to other methods generating large volume EM data, such as serial block-face SEM (SBF-SEM) or focused ion beam SEM (FIB-SEM), this approach has two major advantages: (1) The resin-embedded sample is conserved, albeit in a sliced-up version. It can be stained in different ways and imaged with different resolutions. (2) As the sections can be post-stained, it is not necessary to use samples strongly block-stained with heavy metals to introduce contrast for SEM imaging or render the tissue blocks conductive. This makes the method applicable to a wide variety of materials and biological questions. Particularly prefixed materials e.g., from biopsy banks and pathology labs, can directly be embedded and reconstructed in 3D.

This protocol targets specific cells in tissue for imaging at nanoscale resolution using a scanning electron microscope (SEM). Large numbers of serial sections from resin-embedded biological material are first imaged in a light microscope to identify the target and then in a hierarchical manner in the SEM.

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 system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

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 than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

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 liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

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 disease. To study the formation of PAAs, we developed a protocol using whole-mount immunofluorescence coupled with benzyl alcohol/benzyl benzoate (BABB) tissue clearing, and confocal microscopy. This allows for the visualization of the pharyngeal arch endothelium at a fine cellular resolution as well as the 3D connectivity of the vasculature. Using software, we have established a protocol to quantify the number of endothelial cells (ECs) in PAAs, as well as the number of ECs within the vascular plexus surrounding the PAAs within pharyngeal arches 3, 4, and 6. When applied to the whole embryo, this methodology provides a comprehensive visualization and quantitative analysis of embryonic vasculature.

Here, we describe a protocol to visualize and analyze the pharyngeal arch arteries 3, 4, and 6 of mouse embryos using whole-mount immunofluorescence, tissue clearing, confocal microscopy, 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 is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans has emerged as one of the best models for studying development. From an optical point of view, its size and transparent body make this nematode an ideal specimen for DIC (Differential Interference Contrast or Nomarski) microscopy. This article illustrates a protocol for growing C. elegans nematodes, preparing and mounting their embryos, performing 4D microscopy and cell lineage tracing. The method is based on multifocal time-lapse records of Nomarski images and analysis with specific software. This technique reveals embryonic developmental dynamics at the cellular level. Any embryonic defect in mutants, such as problems in spindle orientation, cell migration, apoptosis or cell fate specification, can be efficiently detected and scored. Virtually every single cell of the embryo can be followed up to the moment the embryo begins to move. Tracing the complete cell lineage of a C. elegans embryo by 4D DIC microscopy is laborious, but the use of specific software greatly facilitates this task. In addition, this technique is easy to implement in the lab. 4D microscopy is a versatile tool and opens the possibility of performing an unparalleled analysis of embryonic development.

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

Here, we present a protocol for preparing and mounting Caenorhabditis elegans embryos, recording development under a 4D microscope and tracing cell lineage.

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