Summary

Rápida aquisição de imagens 3D usando de alta resolução episc Microscopia

Published: November 21, 2016
doi:

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

Todas as utilizações de animais são aprovados pelo Comitê Cuidado e Uso Institucional Animal do Hospital Infantil de Boston. Preparação 1. Amostra acasalamento cronometrada Coloque um C57BL6 tipo selvagem macho e uma fêmea juntos na mesma gaiola. Verifique para a formação de um tampão genital cedo na manhã seguinte. A ficha de acasalamento (aka, vaginal ou a ficha cópula) é uma deposição gelatinosa endurecido que bloqueia…

Representative Results

Nós relatamos imagens HREM alta qualidade de embriões de rato entre E9.5 e E13.5 8. A qualidade de imagem dos embriões final faseada, no entanto, é significativamente comprometida devido à penetração nos tecidos limitada do eosina corante fluorescente. Para aumentar a eficiência de coloração, testámos vários métodos de pré-tratamento que são compatíveis com imagens fluorescentes 11. Especificamente, os embriões de camundongos E15.5 foram tratados co…

Discussion

Aqui, apresentamos um protocolo modificado utilizando equipamentos de laboratório de rotina para obter imagens HREM de série que são compatíveis para uma rápida visualização 3D e análises morfométricas de estruturas complexas. Porque as imagens de alta resolução são tomadas diretamente da face do bloco, em vez de seções individuais, as características morfológicas finas são preservados e rapidamente reconstruída digitalmente em um programa de visualização 3D.

imagens 3D o…

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

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Cite This Article
Zhang, H., Huang, J., Liu, X., Zhu, P., Li, Z., Li, X. Rapid Acquisition of 3D Images Using High-resolution Episcopic Microscopy. J. Vis. Exp. (117), e54625, doi:10.3791/54625 (2016).

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