Applicability, usability, and limitations of murine embryonic imaging with optical coherence tomography and optical projection tomography Manmohan Singh,1 Raksha Raghunathan,1 Victor Piazza,2 Anjul M. Davis-Loiacono,3 Alex Cable,3 Tegy J. Vedakkan,2 Trevor Janecek,1 Michael V. Frazier,1 Achuth Nair,1 Chen Wu,1 Irina V. Larina,2 Mary E. Dickinson,2 and Kirill V. Larin1,2,4,* 1
Department of Biomedical Engineering, University of Houston, 3605 Cullen Boulevard, Houston, 77204, USA Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, 77584, USA 3 Thorlabs, Inc., 56 Sparta Ave., Newton, 07860, USA 4 Department of Electrical Engineering, Samara National Research University, Samara, 34 Moskovskoye sh., 443086, Russia * [email protected] 2
Abstract: 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 highresolution 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 OCIS codes: (110.4500) Optical coherence tomography; (110.6955) Tomographic imaging; (170.3880) Medical and biological imaging.
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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 ModeLocked 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 #261528 (C) 2016 OSA
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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
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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 3013-37-000, 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.
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Fig. 2. OPT system schematic. Table 1. Summary of OPT system optical parameters.
Magnification
Min Zoom 0.39X
Max Zoom 4.9X
0.014
0.072
2.90 mm
0.11 mm
Numerical Aperture Depth of Field Working Distance
114 mm
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 backprojection 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 Embryo Stage E9.5 E11.5 E13.5
Camera Exposure Time (ms) 1800 320 510
Camera gain 5X 3X 2X
Pixel size (µm) 1.97 5.78 7.27
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:
#261528 (C) 2016 OSA
Received 24 Mar 2016; revised 9 May 2016; accepted 10 May 2016; published 19 May 2016 1 June 2016 | Vol. 7, No. 6 | DOI:10.1364/BOE.7.002295 | BIOMEDICAL OPTICS EXPRESS 2301
4 V = π a2c 3
(1)
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
#261528 (C) 2016 OSA
Received 24 Mar 2016; revised 9 May 2016; accepted 10 May 2016; published 19 May 2016 1 June 2016 | Vol. 7, No. 6 | DOI:10.1364/BOE.7.002295 | BIOMEDICAL OPTICS EXPRESS 2302
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. Embryo Stage E9.5 E11.5 E13.5
SNR (dB) OCT 27 26 26
OPT 38 52 51
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 rolloff, but there is a noticeable increase in the noise at longer optical path differences.
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Received 24 Mar 2016; revised 9 May 2016; accepted 10 May 2016; published 19 May 2016 1 June 2016 | Vol. 7, No. 6 | DOI:10.1364/BOE.7.002295 | BIOMEDICAL OPTICS EXPRESS 2303
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.
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Received 24 Mar 2016; revised 9 May 2016; accepted 10 May 2016; published 19 May 2016 1 June 2016 | Vol. 7, No. 6 | DOI:10.1364/BOE.7.002295 | BIOMEDICAL OPTICS EXPRESS 2304
Fig. 6. (a-c) OCT and (d-f) OPT images of the same E9.5 murine embryo (see Visualization 1).
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.
#261528 (C) 2016 OSA
Received 24 Mar 2016; revised 9 May 2016; accepted 10 May 2016; published 19 May 2016 1 June 2016 | Vol. 7, No. 6 | DOI:10.1364/BOE.7.002295 | BIOMEDICAL OPTICS EXPRESS 2305
Fig. 7. (a-d) OCT and (e-h) OPT images of the E11.5 embryo (see Visualization 2).
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. 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, defocusing, and saturation artifacts that reduce image quality.
#261528 (C) 2016 OSA
Received 24 Mar 2016; revised 9 May 2016; accepted 10 May 2016; published 19 May 2016 1 June 2016 | Vol. 7, No. 6 | DOI:10.1364/BOE.7.002295 | BIOMEDICAL OPTICS EXPRESS 2306
Fig. 8. The same E13.5 murine embryo as imaged by the (a-d) OCT and (e-h) OPT systems (see Visualization 3).
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
Department of Biomedical Engineering, University of Houston, 3605 Cullen Boulevard, Houston, 77204, USA Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, 77584, USA 3 Thorlabs, Inc., 56 Sparta Ave., Newton, 07860, USA 4 Department of Electrical Engineering, Samara National Research University, Samara, 34 Moskovskoye sh., 443086, Russia * [email protected] 2
Abstract: 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 highresolution 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 OCIS codes: (110.4500) Optical coherence tomography; (110.6955) Tomographic imaging; (170.3880) Medical and biological imaging.
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61. L. Chen, J. McGinty, H. B. Taylor, L. Bugeon, J. R. Lamb, M. J. Dallman, and P. M. French, “Incorporation of an experimentally determined MTF for spatial frequency filtering and deconvolution during optical projection tomography reconstruction,” Opt. Express 20(7), 7323–7337 (2012). 62. E. A. Susaki, K. Tainaka, D. Perrin, H. Yukinaga, A. Kuno, and H. R. Ueda, “Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging,” Nat. Protoc. 10(11), 1709–1727 (2015). 63. A. Bassi, L. Fieramonti, C. D’Andrea, M. Mione, and G. Valentini, “In vivo label-free three-dimensional imaging of zebrafish vasculature with optical projection tomography,” J. Biomed. Opt. 16(10), 100502 (2011). 64. Z. Zhi, W. Qin, J. Wang, W. Wei, and R. K. Wang, “4D optical coherence tomography-based micro-angiography achieved by 1.6-MHz FDML swept source,” Opt. Lett. 40(8), 1779–1782 (2015). 65. I. V. Larina, K. V. Larin, M. E. Dickinson, and M. Liebling, “Sequential Turning Acquisition and Reconstruction (STAR) method for four-dimensional imaging of cyclically moving structures,” Biomed. Opt. Express 3(3), 650– 660 (2012). 66. M. W. Jenkins, F. Rothenberg, D. Roy, V. P. Nikolski, Z. Hu, M. Watanabe, D. L. Wilson, I. R. Efimov, and A. M. Rollins, “4D embryonic cardiography using gated optical coherence tomography,” Opt. Express 14(2), 736– 748 (2006). 67. T. Inouye, “Image-Reconstruction with Limited Angle Projection Data,” IEEE Trans. Nucl. Sci. 26(2), 2665– 2669 (1979). 68. E. Y. Sidky, C. M. Kao, and X. H. Pan, “Accurate image reconstruction from few-views and limited-angle data in divergent-beam CT,” J. XRay Sci. Technol. 14, 119–139 (2006). 69. M. E. Davison, “The Ill-Conditioned Nature of the Limited Angle Tomography Problem,” SIAM J. Appl. Math. 43(2), 428–448 (1983). 70. A. H. Andersen, “Algebraic reconstruction in CT from limited views,” IEEE Trans. Med. Imaging 8(1), 50–55 (1989). 71. W. Drexler, M. Liu, A. Kumar, T. Kamali, A. Unterhuber, and R. A. Leitgeb, “Optical coherence tomography today: speed, contrast, and multimodality,” J. Biomed. Opt. 19(7), 071412 (2014).
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 ModeLocked 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 #261528 (C) 2016 OSA
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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
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Received 24 Mar 2016; revised 9 May 2016; accepted 10 May 2016; published 19 May 2016 1 June 2016 | Vol. 7, No. 6 | DOI:10.1364/BOE.7.002295 | BIOMEDICAL OPTICS EXPRESS 2299
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 3013-37-000, 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.
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Received 24 Mar 2016; revised 9 May 2016; accepted 10 May 2016; published 19 May 2016 1 June 2016 | Vol. 7, No. 6 | DOI:10.1364/BOE.7.002295 | BIOMEDICAL OPTICS EXPRESS 2300
Fig. 2. OPT system schematic. Table 1. Summary of OPT system optical parameters.
Magnification
Min Zoom 0.39X
Max Zoom 4.9X
0.014
0.072
2.90 mm
0.11 mm
Numerical Aperture Depth of Field Working Distance
114 mm
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 backprojection 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 Embryo Stage E9.5 E11.5 E13.5
Camera Exposure Time (ms) 1800 320 510
Camera gain 5X 3X 2X
Pixel size (µm) 1.97 5.78 7.27
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:
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Received 24 Mar 2016; revised 9 May 2016; accepted 10 May 2016; published 19 May 2016 1 June 2016 | Vol. 7, No. 6 | DOI:10.1364/BOE.7.002295 | BIOMEDICAL OPTICS EXPRESS 2301
4 V = π a2c 3
(1)
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
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Received 24 Mar 2016; revised 9 May 2016; accepted 10 May 2016; published 19 May 2016 1 June 2016 | Vol. 7, No. 6 | DOI:10.1364/BOE.7.002295 | BIOMEDICAL OPTICS EXPRESS 2302
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. Embryo Stage E9.5 E11.5 E13.5
SNR (dB) OCT 27 26 26
OPT 38 52 51
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 rolloff, but there is a noticeable increase in the noise at longer optical path differences.
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Received 24 Mar 2016; revised 9 May 2016; accepted 10 May 2016; published 19 May 2016 1 June 2016 | Vol. 7, No. 6 | DOI:10.1364/BOE.7.002295 | BIOMEDICAL OPTICS EXPRESS 2303
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.
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Received 24 Mar 2016; revised 9 May 2016; accepted 10 May 2016; published 19 May 2016 1 June 2016 | Vol. 7, No. 6 | DOI:10.1364/BOE.7.002295 | BIOMEDICAL OPTICS EXPRESS 2304
Fig. 6. (a-c) OCT and (d-f) OPT images of the same E9.5 murine embryo (see Visualization 1).
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.
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Received 24 Mar 2016; revised 9 May 2016; accepted 10 May 2016; published 19 May 2016 1 June 2016 | Vol. 7, No. 6 | DOI:10.1364/BOE.7.002295 | BIOMEDICAL OPTICS EXPRESS 2305
Fig. 7. (a-d) OCT and (e-h) OPT images of the E11.5 embryo (see Visualization 2).
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. 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, defocusing, and saturation artifacts that reduce image quality.
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Received 24 Mar 2016; revised 9 May 2016; accepted 10 May 2016; published 19 May 2016 1 June 2016 | Vol. 7, No. 6 | DOI:10.1364/BOE.7.002295 | BIOMEDICAL OPTICS EXPRESS 2306
Fig. 8. The same E13.5 murine embryo as imaged by the (a-d) OCT and (e-h) OPT systems (see Visualization 3).
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
