Noninvasive Functional and Structural Imaging of Embryonic Development

Abnormalities in the growth and development of the cardiovascular (CV) system are the most common class of congenital birth defects and the leading cause of birth defect related deaths. Since the heart is formed and begins to beat in humans before it can be imaged by clinical ultrasound, we know very little about early heart dynamics and function in humans. Embryologists rely heavily on animal models to understand the etiology of human birth defects. The mouse is an excellent model to study aberrant cardiovascular development and over the past 10 years, there have been astounding developments in mouse genomics to saturate the genome with mutations and to identify genes with novel roles in CV development and disease. Now there is a pressing need for better tools for phenotyping mutant embryos to reveal primary, early defects that lead to cardiac failure and long-term disorders. Although Ultrasound has been routinely used, mouse embryos are half a millimeter when the heart begins to beat so new tools such as Optical Coherence Tomography (OCT) that offer significant advantages in spatial resolution and sensitivity are critically needed. The OCT is an excellent compromise between ultrasound and confocal microscopy, offering millimeters of depth penetration and high spatial (2-10um lateral) and temporal resolution. An example of OCT and high-frequency ultrasound images of the same embryonic head (E14.5) are shown in Fig 1.

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Fig 1: Comparison between images obtained using (a) OCT and (b) high-frequency ultrasound of the same E14.5 embryo. Scale bar, 500µm.

Recently we obtained encouraging results that demonstrate the capability of OCT for structural imaging of embryonic heart during critical stages of development (Fig 2). As one can see from the image, the internal structures of the heart as well as other details of the embryo are clearly outlined on the images. The spatial resolution that can be achieved by this method provides excellent detail of many easily recognizable structural features, as labeled. For instance, the amnion labeled in A and D is a single cell layer thick and is clearly visible, even in very early 7.5 dpc embryos. It is also possible in these images to see the cell layers that form the trabeculae of the heart ventricle (visible as lines through the heart in A, B). Thus, we are able to produce beautiful OCT structural images of the heart.

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Fig 2: Structural imaging of early embryos with SS-OCT. (A) Live imaging of 8.5 dpc embryonic heart at 30 fps. B, C) 3-D reconstructions of 9.5 dpc embryo with and without the yolk sac, respectively. D) 3-d reconstruction of a 7.5 dpc embryo. The acquisition was performed at 512 A-scans per frame (without averaging, which resulted in the acquisition rate of 31 frames per second).

A 4-D representation of the beating embryonic heartbeat is shown in Fig 3. Figure 3A shows a selection of frames corresponding to different Y positions after de-noising and synchronization, and Figure 3B is a cross-section view of the same reconstruction. This result is the first 4-D reconstruction of mammalian embryonic heartbeat in live embryo culture with OCT. The reconstruction provides sufficient details about tissue structure and permit tracking of the heart wall movements during the cardiac cycle.

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Fig 3:  4-D reconstruction of embryonic cardio dynamics. (A) A gallery of denoised and synchronized time lapses acquired at different y positions and used for 4-D reconstruction of the heartbeat. (B) Corresponding 4-D reconstruction in cross-section view: xz (central), yz (right), and xy (bottom).

Figure 4 shows an example of OCT Doppler measurements of blood flow velocity in 9.5 day. Strong Doppler signals were detected from two regions in the shown field of view (Figure 4B): a yolk sac vessel (blue, labeled 1) and the dorsal aorta (red, labeled 2). The velocity profiles across the vessel have a parabolic shape, as expected.  The peak flow velocity across the vessel was plotted vs. time (Figure 4D) and reveals the periodicity of the cardiac cycle, which can be used to calculate the heart rate of the embryo.

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Fig 4: SS-OCT Doppler measurement of blood flow in the live embryo culture. (A) Structural image of 9.5 dpc embryo showing a fragments of a yolk sac (ys) and an embryonic trunk (et). (B) Corresponding color coded Doppler image showing strong signals from the yolk sac vessel (1) and a dorsal aorta (2). (C) Blood flow velocity profiles measured along the line shown in (A) at different phases of the heart beat cycle. (D) Dynamics of the peak blood flow velocity during the heartbeat. The angle between the direction of the flow and the scanning beam required for the velocity calculation was determined from the 3-d reconstruction of the vessel orientation.

Optical projection tomography (OPT) is a relatively new technique developed to fill a gap in high throughput and high resolution 3D imaging of samples 1 to 10 mm in size. OPT is an optical analog of micro-CT and, similarly, utilizes back-projection for 3D reconstruction of the sample. OPT can provide label-free structural images with exquisite detail by capturing tissue autofluorescence. Figure 5 and corresponding visualization illustrates the same E11.5 mouse embryo as imaged by the OCT and OPT systems. External structures such as the limb buds and eye are easily visible from the OCT images. The OCT sagittal and coronal slices reveal major internal structures such as the mesencephalic vesicle and third and fourth vesicles.The OCT coronal slice also demonstrates the limited penetration depth. OPT overcomes this problem (though clearing is required - thus no life imaging is possible). From the OPT 3D reconstruction, the eye, limb buds, tail, and somites are visible. The OPT sagittal and coronal slices show internal structures such as the pericardial and peritoneal cavities, and fine structures such as Rathke’s pouch were also imaged. 

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Fig 5: (a-d) OCT and (e-h) OPT images of the E11.5 embryo.