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We present an analysis of imaging murine embryos at various embryonic developmental stages (embryonic day 9.5, 11.5, and 13.5) by optical coherence tomography (OCT) and optical projection tomography (OPT). We demonstrate that while OCT was capable of rapid high-resolution live 3D imaging, its limited penetration depth prevented visualization of deeper structures, particularly in later stage embryos. In contrast, OPT was able to image the whole embryos, but could not be used in vivo because the embryos must be fixed and cleared. Moreover, the fixation process significantly altered the embryo morphology, which was quantified by the volume of the eye-globes before and after fixation. All of these factors should be weighed when determining which imaging modality one should use to achieve particular goals of a study.
© 2016 Optical Society of America
1. Introduction
Murine models have provided enormous insights into the mechanisms that underlie mammalian development and birth defects. While genetic profiles and subsequent databases of disease markers have been immensely valuable, phenotypic information is often just as important but is not as well documented [1–4]. The development of new 3D imaging tools now offers new ways to characterize and visualize structural abnormalities.
Analysis of mammalian embryo development has traditionally been done via histological sectioning. The development and adaptation of imaging techniques such as ultrasound biomicroscopy (UBM) [5, 6], micro X-ray computed tomography (micro-CT) [6, 7], and micro magnetic resonance imaging (micro-MRI) [8, 9], has proven invaluable at providing phenotypic information of small mammalian embryos noninvasively. However, each of these techniques has limitations. For example, UBM can provide high spatial resolution (< 100 µm), but image quality depends heavily on the operator. Moreover, the frequencies required to achieve these resolutions result in poor tissue contrast, limited penetration depth, and blood backscatter artifacts [10]. Micro-CT can also provide high spatial resolution (< 100 µm), but the effects of the ionizing radiation can be deleterious, especially for longitudinal analysis. On the other hand, micro-MRI does not rely on ionizing radiation and can provide high spatial resolution (< 50 µm) and superior tissue contrast, but requires high field strengths (≥ 7 T) for detailed images without the use of contrast agents. Recently, in utero micro-MRI has been performed at the cost of spatial resolution, and the gating and registration methods required to remove maternal motion resulted in extended acquisition times of ~2 hours [11].
Optical coherence tomography (OCT) is a well-established noninvasive imaging modality based on low coherence interferometry which provides µm spatial resolution [12]. OCT has been successfully used in clinical applications such as ophthalmology [13, 14] and cardiology [15, 16] and research applications such as cancer imaging [17, 18] and developmental biology [19–21]. With the development of faster optical sources such as Fourier Domain Mode-Locked Lasers [22–24] and graphics processing unit (GPU) accelerated software [25], OCT is now capable of providing high resolution real-time video-rate 3D acquisition and visualization. However, whole-body imaging of murine embryos at later stages with OCT is still a challenge due to the limited penetration depth. Techniques such as optical clearing can ameliorate this limitation [26], but clearing applications in murine embryonic imaging have been very scarce and optical clearing is generally not compatible with live samples [27, 28]. We have recently developed a multi-angle OCT imaging technique to image deeper embryonic structures [29]. However, only embryos up to 10.5 days post-coitum were imaged, and the technique is sensitive to translation errors and requires accurate rescaling according to the sample refractive index. Nevertheless, OCT is widely used for murine embryonic imaging due to its noninvasive nature, which enables in utero imaging, imaging of embryos in culture, and subsequent longitudinal investigations [30]. In addition to structural analysis, techniques such as Doppler [31], speckle variance [32], and spectroscopy [33] can provide additional information for applications such as blood flow analysis [31, 34] and microvasculature imaging [35–37].
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 [38, 39]. 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 and can also be used to image fluorescence labeled targets for spatio-temporal functional imaging and genotypic analysis [40–43]. However, murine embryonic samples require lengthy fixation, clearing, and immobilization procedures, hindering OPT applications for live murine imaging. Whole-body in vivo OPT imaging of samples which are small, such as D. melanogaster [44], or transparent such as C. elegans [45] and D. rerio [46], has been reported. While OPT has been used for imaging live murine embryos with the development of life support systems and motion artifact compensation, only a limb bud was imaged [47, 48]. Furthermore, due to the back-projection reconstruction procedure, OPT cannot be used for real-time viewing. Nevertheless, the incorporation of GPU acceleration has drastically reduced the reconstruction time [49].
There has currently been no direct comparison of OCT and OPT for murine embryonic developmental imaging. To fill this gap, we have imaged embryos of various (E) embryonic developmental stages (9.5, 11.5, and 13.5 days post-coitus) with OCT both in vitro and in vivo and then fixed and cleared the same embryos for subsequent OPT imaging. To provide a quantitative basis of the changes in embryo morphology due to the fixation and clearing procedure, the eye volumes were measured by both systems.
2. Material and methods
2.1 Embryo preparation and imaging
For direct comparison of OCT and OPT imaging in vitro, CD-1 murine embryos (Charles River Laboratories, Wilmington, MA, USA) of the three embryonic developmental stages were dissected out, removed from their yolk sacs, placed in a standard culture dish, and immersed in Dulbecco’s Modified Eagle Media (DMEM). The embryos were then imaged by the OCT system, also while immersed in DMEM. After the OCT imaging was completed, the embryos were immediately prepared for OPT imaging. The embryos were fixed in a solution of 4% paraformaldehyde for 2 hours at 4°C on a nutator. The fixed embryos were washed with 1X PBS three times and then mounted in 1% agar. Excess agar was cut away and the agar blocks containing the embryos were dehydrated by immersion in increasing concentrations of methanol (25%, 50%, 75%, 100%, 100%, 100% v/v, 2 hours at each step). The embryos were then cleared in benzyl alcohol-benzyl benzoate (BABB) overnight or until sufficiently clear. The agar blocks were mounted with glue on a magnetic sample chuck, which was magnetically attached to a rotational stage. During OPT imaging, the embryos were immersed in BABB in a square glass cuvette to enhance optical clearing, reduce background autofluorescence, and ensure a relatively uniform sample refractive index.
For live OCT imaging (in vivo), E9.5 embryos were dissected out and placed in the DMEM culture media while still within the yolk sac. The embryos were given at least 30 mins to recover from the extraction procedure in a 37°C incubator with 5% CO2 [37]. After the recovery period, the embryos were imaged, also while immersed in the DMEM culture solution. All OCT embryo imaging was performed with the OCT system sample arm and embryos placed in the incubator [50].
All procedures were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee and adhered to its animal manipulation policies.
2.2 Optical coherence tomography (OCT) system
The commercial OCT system was composed of a swept source laser with an A-scan rate of 200 kHz, central wavelength of ~1300 nm, bandwidth of ~100 nm, and sample incident power of ~12 mW (Model OCS1310V2, Thorlabs Inc, NJ, USA). A schematic of the OCT system can be seen in Fig. 1. A-scan averaging was utilized during all structural imaging to reduce background noise and enhance SNR. The sample arm of the OCT system was placed in the incubator to ensure viability of the embryos during live imaging. The images were corrected to physical dimensions assuming that the refractive index of the media and embryos was 1.4 [37].
Fig. 1 OCT system schematic. BPD: balanced photodetector; PC: polarization controller; C: collimator; VA: variable attenuator; RM: reference mirror; DM: dichroic mirror.
2.3 Optical projection tomography (OPT) system
A home-built OPT system, based on a published design [51] with slight modifications and adaptations, was composed of three main components: illumination sources, sample stage, and microscope, as shown in Fig. 2. The illumination sources were a white light source (to aid with alignment) and a broadband excitation source (X-Cite Exacte, Excelitas Technologies Corp., MA, USA) in conjunction with an excitation filter (482 ± 17 nm for autofluorescence). The sample stage was comprised of two motorized linear stages (X and Y axes in Fig. 2), for aligning the sample within the microscope field of view, and a motorized rotational stage. The microscope stage was composed of an emission filter (593 ± 20 nm for autofluorescence), a 0.75X objective lens (P/N 29-20-39-0000, Qioptiq, MA, USA), a fine focus module (P/N 30-13-37-000, Qioptiq, MA, USA), zoom module (P/N 30-61-38-000, Qioptiq, MA, USA), 1.0X optical relay (TV) tube (P/N 29-90-72-000, Qioptiq, MA, USA), and CCD camera (Retiga 4000DC, QImaging, BC, Canada). The optical parameters of the system at the minimum and maximum zoom are presented in Table 1. While the CCD was capable of acquiring projections at 2048x2048 pixels, the imaging field of view was set to 1536x1536 pixels due to vignetting.
Table 1. Summary of OPT system optical parameters.
Before imaging, the sample was aligned to ensure that rotation occurs around the sample vertical axis to prevent reconstruction artifacts. The sample was raised out of the BABB solution and a series of dark-field images were taken and averaged. Once the sample was lowered back into the BABB solution, a 2D projection was captured every 0.3° over the full 360° of rotation for a total of 1200 images. The averaged dark-field was subtracted from each of the projections and then the samples were reconstructed by standard filtered back-projection in NRecon (Bruker microCT, Belgium). The incident power on the sample was ~68 mW, and the camera gain and exposure time were selected such that the dynamic range of the real-time projection of the sagittal plane was maximized. A brief summary of the acquisition parameters for each of the embryo stages is provided in Table 2.
Table 2. Summary of OPT acquisition parameters
2.4 Quantification of eye volumes
The effects of dehydration after fixation of embryos for sectioning and imaging with electron microscopy have been previously studied [52, 53], but there has been no direct quantification of the effects of the clearing, fixation, dehydration, and immobilization procedure for OPT imaging on murine embryonic morphology. To quantify this effect, the eye volumes of the embryos were quantified with our previously published technique [54]. Briefly, the eye was modeled as an oblate spheroid [55], and the minor (along the optical axis) and major (orthogonal to the optical axis) axes lengths were obtained from the scaled OCT and OPT images. The volume was calculated by:
where a and c were the lengths of the major and minor axes, respectively.3. Results
3.1 OCT and OPT system performance
To provide experimentally measured transverse resolutions, a US Air Force resolution target was imaged by both systems, as shown in Fig. 3. The image of the central region of the resolution target as obtained by the OCT system is shown in Fig. 3(a). Figure 3(b) is the yellow outlined region in Fig. 3(a) and shows that group 5, element 5 was the smallest fully and clearly resolved line group, which corresponded to a line width of ~10 µm. Some anisotropic characteristics can be seen, which is why the worst resolution of the two directions of line groups was selected. The intensity profile of the yellow line drawn in Fig. 3(b) is plotted in Fig. 3(c). Figure 3(d) is the image of the resolution target obtained by the OPT system at maximum zoom. Figure 3(e) is an magnified view of the highlighted region in Fig. 1(d) and shows that the smallest fully resolved line group was group 7, element 2, which corresponded to a line width of ~3.5 µm. Figure 3(f) shows the transverse intensity profile of the line drawn in Fig. 3(e). Therefore, the transverse resolution was ~10 µm for the OCT system and ~3.5 µm for the OPT system at maximum zoom.
Fig. 3 Transverse resolutions of the (a-c) OCT and (d-f) OPT systems as determined by a US Air Force resolution target. (a,d) View of the resolution target, (b,e) magnified region which is outlined in (a,d), and (c,f) transverse intensity profile of the yellow line in (b,e) with a line width of ~16 µm and ~3.5 µm, respectively.
Figure 4 shows the axial resolution measurements for the OCT and OPT systems. The axial resolution of the OCT system was determined by imaging a mirror, which is depicted in Figs. 4(a), 4(b). Figure 4(b) plots an axial intensity profile of a single A-line of the mirror in Fig. 4(a). The width at the −3 dB corners was used to determine that the axial resolution of the OCT system was ~12 µm. The axial resolution of the OPT system was experimentally determined by imaging a 5 µm polystyrene microsphere embedded in agarose, which can be seen in Figs. 4(c), 4(d). A tungsten rod was also embedded to provide a reference for the axis of rotation after the image was reconstructed by back-projection. From the reconstructed image, a transverse intensity profile that bisected the microsphere and was co-linear with a diameter of the rod was selected for determining the axial resolution of the OPT system as illustrated by the yellow line in Fig. 4(c). The FWHM of the axial intensity profile, which is the yellow line in Fig. 4(d), was ~7.5 µm (at ~72% of the maximum stepper motor zoom). Therefore, the axial resolutions of the OCT and OPT system were determined to be ~12 µm and ~7.5 µm, respectively.
Fig. 4 Axial resolutions of the (a,b) OCT system and (c-e) OPT system. (a) OCT image of a mirror and (b) axial intensity profile of the yellow line in (a). The width at the −3dB corners was ~12 µm. (c) OPT reconstruction of tungsten rod, 5 µm microsphere embedded in agar, and extrapolated diameter of the rod used for obtaining the intensity profile of the microsphere in the axial direction. (d) Zoomed in view of the microsphere highlighted in (c). (e) Intensity profile of the yellow line in (d), where the FWHM was ~7.5 µm.
The signal-to-noise ratio (SNR) of the systems was also evaluated on the embryo images to provide a quantitative metric of system performance. A region of interest (ROI) of the embryos was chosen and the SNR was calculated as SNR = 20•log10(µROI/σbackground), where µROI was the mean of the signal within the ROI and σbackground was the standard deviation within the ROI. The ROI was an area with a bright signal within the embryo tissue, so the SNR shows a best case scenario. Both the OCT and OPT images were scaled in dB before SNR calculation to provide a direct comparison, and Table 3 provides a summary of the SNR performance of the systems for embryo imaging.
Table 3. SNR of the OCT and OPT systems calculated from the embryo images.
Additionally, the OCT system sensitivity and sensitivity roll-off were experimentally evaluated. A mirror was placed at the focal plane of the OCT sample arm and the reference arm was translated every 500 µm. An attenuation filter was placed between the mirror and OCT scan objective to attenuate the light at 74 dB (measured as double path). Figure 5 plots single axial scans while the reference mirror was translated. The sensitivity (peak intensity/mean of the local noise) for each optical path difference after addition of the double path attenuation [56] is noted on the Fig. 5. The noise was averaged from the intensity values from 1 mm surrounding the mirror. The system shows an almost negligible sensitivity roll-off, but there is a noticeable increase in the noise at longer optical path differences.
Fig. 5 OCT axial scans from an image of a mirror with 74 dB attenuation (double pass) filter in the sample arm. The reference arm was translated at 500 µm increments. The sensitivity after addition of the attenuation is noted for each optical path difference.
3.2 Murine embryos
Multiple views of the embryos at different developmental stages as imaged by the OCT system and OPT system are displayed in the figures below to directly compare the two imaging systems. In order to eliminate inter-sample variability, images from the same embryos at each developmental stage as imaged by both systems are displayed. It should be noted that the embryos prepared for OPT imaging are smaller as the fixation, dehydration, and clearing processes significantly shrank the embryos. Each Visualization for the respective embryonic development stages shows a 3D rotation of the embryo as imaged by the (left) OCT and (right) OPT systems, followed by slicing through the sagittal plane.
3.2.1 E9.5 embryos
Figure 6 and Visualization 1 shows OCT and OPT images of E9.5 embryo. The data demonstrate that both systems were able to provide whole body images of the embryos at this developmental stage. However, from the OCT images, only large external structures such as the tail and limb buds can be clearly seen. Furthermore, the limited imaging depth of OCT is clearly illustrated by the coronal slices, and shadows caused by the tail and limb buds occlude visualization of internal organs. Nevertheless, the sagittal slices show that some internal structures, such as the telencephalic vesicle, aortic sac, and peritoneal cavity were still well resolved. In addition, the OCT coronal slice shows the formation of smaller structures such as the optic cup.
The OPT 3D reconstruction demonstrates that external structures such as the tail, mouth, and otic vesicle were clearly imaged. Furthermore, the sagittal and coronal slices show that the OPT system was able to reveal the formation of structures such as the sinus venosus, aortic sac, and branchial arch arteries.
3.2.2 E11.5 embryos
Figure 7 and Visualization 2 illustrates the same E11.5 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 and that the OCT system was unable to wholly image the E11.5 embryo.
From the OPT 3D reconstructions, 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. However, saturation of the internal organs, vignetting, and blurring at the peripheries resulted in reduced image quality.
3.2.3 E13.5 embryos
Figure 8 and Visualization 3 depict the same E13.5 embryo as imaged by the OCT and OPT systems. The OCT 3D reconstruction shows external structures such as the ear, eye, limbs (including digits), and tail. While the penetration depth is limited, structures such as the ocular lens can still be visualized.
Fig. 8 The same E13.5 murine embryo as imaged by the (a-d) OCT and (e-h) OPT systems (see Visualization 3).
Similar to the OCT reconstruction, the OPT 3D reconstruction also shows the ear, limbs, digits, tail, and eye. In contrast to the OCT system, the internal organs such as the heart, liver, and tongue were clearly imaged by OPT system. Furthermore, fine structures such as the cardiac chambers, incisors, and nasal cavity can also be visualized from the OPT reconstruction. However, similar to the E11.5 data, there is considerable vignetting, de-focusing, and saturation artifacts that reduce image quality.
3.2.4 Live imaging
The major advantage of OCT as compared to OPT is that OCT can be utilized for label-free live imaging. Visualization 4 demonstrates an example of live imaging of an E9.5 embryo. The cardiac contractions can easily be seen, and scatter events from individual erythrocytes show the flow of blood in the yolk sac vasculature and forming heart chambers.
3.2.5 Eye volumes
The eyes in the E9.5 embryos are not developed beyond a simple optic cup, therefore the eye volumes were only quantified for the E11.5 (n = 9) and E13.5 (n = 12) embryos. Figure 9 plots the comparison of the eye volumes, where the error bars are the inter-embryo standard deviation for a given development stage and imaging modality. The eye volumes for the E11.5 embryos were 0.012 ± 0.013 mm3 and 0.005 ± 0.003 mm3 as imaged by OCT and OPT, respectively. The eye volumes for the E13.5 embryos as imaged by the OCT system were 0.052 ± 0.016 mm3, which decreased to 0.021 ± 0.004 mm3 after fixation and clearing for OPT imaging. The differences in eye volumes between imaging modalities, which can also be thought of as before and after the clearing and fixation procedure, for each embryo development stage were very significant (P<0.01 for E11.5 and P<0.0005 for E13.5) by a Mann-Whitney test. The Mann-Whitney test was chosen because all of the data subsets were not normal by a Shapiro-Wilk test with a significance level of 0.05.
Fig. 9 Eye volumes of the E11.5 (n = 9) and E13.5 (n = 12) embryos quantified by modeling the eye-globe as an oblate spheroid and measuring the major and minor axes. Error bars represent the inter-embryo standard deviation for the respective embryonic stage and imaging modality. Statistical testing was performed by a Mann-Whitney test.
4. Discussion and conclusion
OCT has widely been used to study the development of murine embryos in utero and in vitro. The limited penetration depth of less than 2 mm in embryonic tissue prevents whole body imaging at later development stages, particularly at stages beyond E10, and provides poor tissue differentiation without processing by methods such as speckle variance [35, 36, 57]. Furthermore, imaging of deeper structures can be difficult without proper orientation of the sample so that the extremities do not occlude the structures of interest. Nevertheless, previous investigations have successfully utilized OCT to study cardiac [20, 58], ocular [50, 54], vascular [36], and neural [59] development in murine embryos as well as visualize microinjections of drugs [60]. Additional methods such as rotational imaging could be combined with OCT to allow multi-angle embryonic imaging [29].
In contrast, OPT allows for acquisition of high resolution full body images of murine embryos with excellent spatial resolution and contrast and with minimal shadowing artifacts due to back-projection reconstruction after multi-angle projection acquisition. Our results showed that the axial resolution of the system did not exactly match the transverse resolution, which may be due to the lack of telecentricity from the zoom lens. Other effects such as multiple scattering and the limited depth of focus may also have contributed to the reduced resolution. A telecentric lens and a different acquisition and reconstruction scheme may help overcome these limitations by placing half of the sample within the depth of focus and modifying the reconstruction algorithm accordingly [39]. Previous work has also shown that spatial filtering and deconvolution can also increase the resolution and image quality [61], and integrating these improvements is a direction of our future work. However, OPT requires lengthy embryo fixation and clearing, hindering its use for live murine embryonic imaging [38, 39]. In addition, the clearing procedure alters the morphology of the embryos as evidenced by the eye volume results in Fig. 8, making quantitative direct comparisons to unaltered embryos difficult [38, 43]. The eye volume results do show that there was a very significant change in the morphology due to the clearing procedure, but the shrinking effect was exaggerated in the eye-globe due to the relatively large amount of fluid in the eye-globe. Regardless, shrinkage of the entire embryo is also noticeable by the differences in the scale bars between imaging modalities of the embryos of all three stages. While the effects of the fixation procedures on embryonic morphology have been previously studied for electron microscopy and sectioning [52, 53], our results show that there is a need for systematic analysis of the OPT preparation procedure to fully understand its effects tissue-wise in order to quantitatively compare OPT results to unaltered embryos. However, more advanced tissue clearing protocols (e.g. CUBIC) have been developed that can better maintain embryo morphology as compared to the BABB clearing procedure [62].
While the results show that the SNR for embryonic imaging of the OPT system is somewhat better than those of the OCT system (Table 1), these measurements were made on a bright region. Improper clearing and blood autofluorescence can lead to poor contrast and reduced image quality in other locations as seen by the bright internal organs of the dissected E11.5 and E13.5 embryos. In addition to saturation, vignetting resulted in a lack of contrast at the peripheries of the E11.5 and E13.5 embryos, which was caused by the combination of insufficient excitation spot size and inadequate internal organ and blood clearing. Moreover, reconstruction and de-focusing artifacts can be seen in the E11.5 and E13.5 caused by misalignment and a limited depth of field, respectively. OPT has been used successfully for in vivo imaging applications of transparent samples such as D. rerio vasculature imaging [63], but live imaging of murine embryos is still a challenge [47]. Nevertheless, our results show that OPT can be a powerful tool for analyzing murine embryonic development in fixed samples.
Most current OCT systems can acquire a volume of a live sample with sufficient spatial resolution in a few seconds. However, recent advances in OCT source hardware have enabled the acquisition of a volume in milliseconds [22, 23], enabling rapid 4D investigations such as micro-angiography [64]. This has enabled our group to study structural and functional murine embryo cardiodynamics at a volume rate of 43 Hz with direct 4D acquisition [58], whereas previous 4D analysis has relied on post-processing reconstruction methods [21, 65, 66]. Naturally, this method of direct 4D acquisition paves the way for 5D (x, y, z, time, development stage) embryonic cardiovascular development investigations without reliance on complex and time consuming post-processing reconstruction with the added benefit of a significantly reduced data size and acquisition time. In contrast, OPT utilizes back-projection and thus, cannot be used for real-time applications. Additionally, OPT can also require extended acquisition times for early stage embryos due to a lack of sufficient tissue that is capable of producing autofluorescence. Long CCD exposure times can lead to acquisitions exceeding 30 minutes, and large gains can exacerbate noise which reduces image quality as particularly seen by the presence of the grain in the E9.5 embryo images in this study. Limited angle acquisition is a viable option for reducing the overall acquisition time. Methods to accurately reconstruct the data from limited angles have been available for decades and have been continuously developed due to the wide clinical adoption of CT imaging [67–70].
Because of the rapid advancement and clinical adoption of OCT, different imaging techniques have been combined with OCT for complementary or supplementary contrast [71], such as various fluorescence and nonlinear microscopy techniques, Raman spectroscopy, coherent anti-Stokes Raman spectroscopy, and photoacoustic imaging. In this manuscript we show the comparison between images of murine embryos made with OCT and then OPT allowing for live imaging with OCT followed by more complete 3D analysis with OPT. While OPT imaging of the live mouse embryo is limited, a combined OCT/OPT system could potentially be useful for weakly scattering samples such as D. rerio, which has been extensively studied using both OCT and OPT. This would potentially enable longitudinal phenotypic and genotypic investigations that would provide a plethora of valuable information for correlating genetic changes with their resultant phenotypic outcomes.
Additionally, OCT/OPT system potentially could be used for studies in which live analysis of blood flow measured from peripheral tissues supplement structural information about cardiac defects. It is often the case that subtle structural defects can be detected in mutant mice but the addition of functional data about blood flow could not only be used to confirm the defect but could also allow for quantitation of the severity of the defect. Moreover, there is a real potential to use OCT-guided injection to label vasculature or other tissues, groups of cells and then analyze with OPT [60]. Outside of mouse embryos, this combination could be useful in, for example, the eye model where blood flow analysis can be combined with a 3D examination of markers for pericytes or vascular pathology to relate these two events.
In summary, here we have shown the applicability of OCT and OPT for imaging murine embryonic development. Embryos of various embryonic development stages (E9.5, E11.5, and E13.5) were first imaged by a commercially-available OCT system. The embryos were then fixed, cleared, and imaged by a home-built OPT system. The OCT system was only able to whole-body image the E9.5 embryos because of the significant light attenuation and exhibited shadowing artifacts from external structures, such as the limbs and tail. However, internal structures such as the brain ventricles and ocular structures were still captured by the OCT system. OCT was also able to perform live imaging to capture the heartbeat of an E9.5 embryo. In contrast, the OPT system was able to wholly image the embryos from all three developmental stages, but showed vignetting due to an insufficient excitation spot size and misalignment and defocusing artifacts. Moreover, preparation for OPT imaging required a lengthy embryo fixing and clearing procedure. We have presented the first quantification of the changes in morphology from the OPT clearing and fixation procedure by measuring the volume of the eye-globes of the embryos, which showed that the OPT preparation procedure significantly shrinks the embryonic organs. This effect must be taken into consideration when comparing murine embryos imaged by OPT to unaltered embryos due to dramatic loss in volume after preparation for OPT imaging.
Acknowledgments
The authors would like to thank Dr. Chih-Wei Hsu with his help in embryo clearing and preparation and Dr. Shang Wang for his help with the OCT experiments. Dr. Hsu and Dr. Wang are both with the Department of Molecular Physiology and Biophysics at Baylor College of Medicine (BCM). We would also like to thank the Optical Imaging and Vital Microscopy core at BCM for use of their hardware and computer work stations. This work was funded in part by NIH HL077187, HL095586, U54 HG006348-S1, T32 HL007676, R01HL120140, and R01HD086765.
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