Adquisición rápida de imágenes en 3D de alta resolución Usando Episcópico Microscopía
Summary
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.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
A continuación, presentamos un protocolo modificado mediante equipos de laboratorio de rutina para adquirir imágenes HREM de serie que son compatibles para la visualización 3D rápida y análisis morfométrico de las estructuras complejas. Debido a que las imágenes de alta resolución se toman directamente de la cara del bloque en lugar de secciones individuales, las características morfológicas finas se conservan y rápidamente reconstruidas digitalmente en un programa de visualización en 3D.
<p class="jove_…Disclosures
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| Mice | The Jackson Laboratory | C57BL6 | |
| Ethyl Alcohol | Pharmco-Aaper | 111000200 | 200 Proof, Absolute, ACS/USP/Kosher Grade |
| Formalin | Sigma | HT501128-4L | |
| Urea | Sigma | U5128 | Clearing Solution |
| Glycerol | Fisher scientific | G33-4 | Clearing Solution |
| Sodium Dodecyl Sulfate | Invitrogen | 15525-017 | Clearing Solution |
| Eosin Y Disodium Salt | Fisher scientific | BP241925 | Infiltration Solution |
| Acridine Orange hemi (zinc chloride) salt | Sigma | A6014 | Infiltration Solution |
| Whatman paper | GE | 3030-153 | |
| JB-4 Embedding Kit – Solution A | Polysciences, Inc. | 0226A-800 | Infiltration Solution |
| JB-4 Embedding Kit – Solution B | Polysciences, Inc. | 0226B-30 | Embedding Solution |
| JB-4 Embedding Kit – Catalyst | Polysciences, Inc. | 02618-12 | Infiltration Solution |
| Embedding Molds | Polysciences, Inc. | 23185-1 | 16x8mm |
| Peel-A-Way Sharp Embedding Molds | Polysciences, Inc. | 18986-1 | 22mm x 22mm square, truncated to 12mm x 12mm |
| JB-4 Plastic Block Holders | Polysciences, Inc. | 15899 | |
| Disposable Graduated Transfer Pipes | VWR | 414004-014 | |
| Petrl Dish | VWR | 25384-088 | Embryo dissection |
| Forceps Inox Tip | ROBOZ | RS-5015 | Embryo dissection |
| Graefe Tissue Forcep | ROBOZ | RS-5153 | Embryo dissection |
| Delicate Operating Scissor | ROBOZ | RS-6700 | Embryo dissection |
| 14 ml Polypropylene Round-Bottom Tubes | Falcon | 352059 | |
| 50 ml Polypropylene Conical Tubes | Falcon | 352098 | |
| Ice Bucket | Magic Touch Iceware International Corp. | R19-343 | |
| 100 mL (3.4 oz.) Antistatic Polystyrene Weigh Boats | VWR | 89106-766 | |
| 330 mL (11.2 oz.) Antistatic Polystyrene Weigh Boats | VWR | 89106-770 | |
| Sodium Chloride | MP Biomedicals | 102892 | PBS Solution |
| Potassium Chloride | Sigma | P0662 | PBS Solution |
| Potassium phosphate monobasic | Sigma | P5405 | PBS Solution |
| Sodium phosphate dibasic heptahydrate | Sigma-Aldrich | S9390 | PBS Solution |
| Digital Camera | Hamamatsu Photonics | C11440-10C | |
| Microtome | Leica | RM2265 | |
| Illuminator | Carl Zeiss | HXP 200C | |
| Fluorescence Stereo Zoom Microscope | Carl Zeiss | Axio Zoom.V16 | |
| Stereo Microscope | Olympus | SZX16 | |
| Fiber Optic Light Source | Leica | KL 1500 LCD | |
| Disposable Microtome Blade | VWR | 95057-832 | |
| Single Edge Dispenser | Personna | 62-0330-0000 | |
| ZEN | Carl Zeiss | pro 2012 | |
| Photoshop | Adobe | CS6 v13.0 | |
| Amira 3D Software | Visage Imaging | 5.4 | |
| Computer | Dell T7600, 2x900GB SAS hard drive and 64 GB DDR3 RDIMM memory | ||
| Rocking Platform Shakers | VWR | 40000-304 | |
| Standard Hot Plate Stirrer | VWR | 12365-382 | |
| Analytical Balance | Mettler Toledo | AB54-S |
References
- Gregg, C. L., Butcher, J. T. Quantitative in vivo imaging of embryonic development: opportunities and challenges. Differentiation. 84, 149-162 (2012).
- Liu, X., Tobita, K., Francis, R. J., Lo, C. W. Imaging techniques for visualizing and phenotyping congenital heart defects in murine models. Birth defects Res C. 99, 93-105 (2013).
- Norris, F. C., et al. A coming of age: advanced imaging technologies for characterising the developing mouse. Trends Genet. 29, 700-711 (2013).
- Weninger, W. J., et al. Phenotyping structural abnormalities in mouse embryos using high-resolution episcopic microscopy. Dis model mech. 7, 1143-1152 (2014).
- Mohun, T. J., Weninger, W. J. Episcopic three-dimensional imaging of embryos. Cold Spring Harb protoc. , 641-646 (2012).
- Sizarov, A., et al. Three-dimensional and molecular analysis of the arterial pole of the developing human heart. J Anat. 220, 336-349 (2012).
- Anderson, R. H., et al. Normal and abnormal development of the intrapericardial arterial trunks in humans and mice. Cardiovasc Res. 95, 108-115 (2012).
- Huang, Y. C., Chen, F., Li, X. Clarification of mammalian cloacal morphogenesis using high-resolution episcopic microscopy. Dev Biol. 409, 106-113 (2016).
- Mohun, T. J., Weninger, W. J. Embedding embryos for episcopic fluorescence image capturing (EFIC). Cold Spring Harb protoc. , 675-677 (2012).
- Weninger, W. J., Mohun, T. J. Three-dimensional analysis of molecular signals with episcopic imaging techniques. Methods mol biol. 411, 35-46 (2007).
- Richardson, D. S., Lichtman, J. W. Clarifying Tissue Clearing. Cell. 162, 246-257 (2015).
- Hama, H., et al. Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat Neurosci. 14, 1481-1488 (2011).
- Susaki, E. A., et al. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell. 157, 726-739 (2014).
- Mohun, T. J., Weninger, W. J. Generation of volume data by episcopic three-dimensional imaging of embryos. Cold Spring Harb protoc. , 681-682 (2012).
