Submicron Imaging of Soft-Tissues Using Low-Dose Phase-Contrast X-Ray Synchrotron Microtomography With an Iodine Contrast Agent
Department of Craniofacial Development and Stem Cell Biology, Dental Institute, King's College London, London, UK
AP-HP, Ho, ̂, pital Pitié-Salpe, ̂, trière, Service de Chirurgie Maxillofaciale et Stomatologie, Paris, France
UPMC Université Paris 06, Paris, France
Correspondence to: RH Khonsari, MD, PhD, AP-HP, Hôpital Pitié-Salpêtriére, Service de Chirurgie Maxillofaciale et Stomatologie, Paris, F-75013, France. Tel:+33685967200, fax:+33145245998. E-mail: firstname.lastname@example.org
3-D visualization of forming organs and tissues in early embryos helps understanding their developmental dynamics. 3-D reconstruction of an organ from an image stack requires: (1) a sufficient number of slices in order to obtain smooth contours, and (2) a satisfactory contrast that allows differentiating between tissue layers during segmentation. Based on these principles, satisfactory but very time-consuming techniques are available for manual segmentation and step-by-step 3-D reconstructions of small embryonic structures using histology (Viriot et al., 1997, 2000).
Usual micro-CT devices available in developmental biology units do not provide the sufficient resolution required to visualize the smaller developing structures at early embryonic stages, such as tooth germs at E11.5. Furthermore, the contrast obtained from soft-tissues is low if high doses of radiation—not supported by small samples—are not used.
A recent study (Raj et al., 2014) has shown that synchrotron imaging with a sliver-based contrast agent provides images of embryonic soft-tissues with a resolution of 4–10 μm. Here we show that the combination of propagation phase contrast, rapid imaging, phase retrieval and iodine contrast agent allows soft-tissue imaging with a voxel size of 0.695 μm and a relatively low radiation dose. This method provides submicronic images where single cells can be individualized.
MATERIAL AND METHODS
CD1 full mouse embryos (stages E11.5 to E13.5) and CD1 mouse embryo heads (stages E14.5 to E17.5) were fixed in 4% PFA overnight and dehydrated gradually up to 100% ethanol. Dehydrated samples were stained overnight using 1% iodine metal in 100% ethanol (Metscher, 2009).
The exact mechanism for contrast enhancement using iodine staining is not understood. Iodine preferentially binds to muscle fibers (and probably bone) compared to connective tissue (Lecker et al., 1997). Contrast enhancement corresponds to an increase of X-ray absorption in selective cell populations, due to a relative excess of entrapped iodine (Jeffery et al., 2011).
Embryos were imaged using the ID19 beamline at the ESRF (European Synchrotron Radiation Facility, Grenoble, France; proposals MD-537 and EC-853). Third generation synchrotrons provide a partially coherent X-ray beam due to the small source size and the long distance between the source and the sample. More precisely, samples were scanned using a 0.678 μm optic coupled to different FreLoN (Fast Readout Low Noise) CCD cameras (Labiche et al., 2007), one adapted for relatively rapid imaging (FreLoN 2K), and the other for high efficiency, but at lower speed (FreLoN E2V). The propagation distance was set at 50 mm to obtain sufficient phase contrast effect in edge detection mode. Half-acquisition (doubling of the field of view by setting the center of rotation on the side of the picture coupled with rotation of the sample over 360°) was applied in local tomography using 3,000 projections of 0.2 s each in continuous rotation mode (Lak et al., 2008). The beam was produced by the U17.6 undulator with a gap closed at 20 mm without filter. The effective energy was 19.2 keV. A single distance phase retrieval process was used (Paganin et al., 2002; Sanchez et al., 2012), coupled to an unsharp mask on the volume (Sanchez et al., 2012). The samples were scanned with isotropic voxel sizes of 0.695 μm (FreLoN 2K full resolution), 0.75 μm (FreLoN E2V full resolution) and 1.4 μm (FreLoN 2K binning). Segmentation and 3-D reconstructions were performed using Mimics 14.0 (Materialise, Leuwen, Belgium).
Full head sections at different stages provided a general view of craniofacial anatomy (Fig. 1a). Both soft and hard tissues were visualized, but the signal from soft tissue structures (muscles, central nervous system, tooth germs) was more informative than the signal from hard tissues, which was very strong and artifacted (mandible) (Figs. 1b–1f).
Cartilage (nasal septum, Meckel's cartilage) could be identified based on the specific aspect of the chondrocytes (Figs. 1b–1d) and easily differentiated from the surrounding mesenchyme. Clear contrast between epithelium and mesenchyme allowed the segmentation of different tissue layers within tooth germs (Figs. 2 and 3).
Cranial nerves and central nervous system were also visualized using synchrotron imaging: the emergence of the optic nerve from the diencephalon could be reconstructed in 3-D (Fig. 3), as well as the oculomotor muscles, which could be reconstructed individually at early stages (Fig. 3b). Red cells emitted a very strong signal, which was of use in localizing blood vessels (internal carotid artery, Fig. 4a and 4b). The smaller anatomical details of the cranial nerves were seen, such as the finer branches of the olfactory nerve at their exit from the nasal cavities (Fig. 4c). In brief, from E11.5 until birth, most anatomical structures were visualized at the cellular level, could be segmented and reconstructed in 3-D, both in full head sections (Fig. 1) and in more focused fields of view (Fig. 2).
In order to provide satisfactory soft-tissues images, usual micro-CT devices used in phase contrast require very high doses of radiations, which would eventually destroy smaller embryos. Using synchrotron light with propagation phase contrast, rapid imaging, phase retrieval and iodine staining of the sample allows to lower the radiation dose and capture cellular-sized structures in early embryos at a submicron resolution: in developmental biology, the smallest relevant forming organs and tissues—such as craniofacial muscles or tooth germs at early developmental stages (Figs. 1 and 2) —can be imaged using this technique. Furthermore, these structures can be readily reconstructed in 3-D after segmentation as early as E11.5 in mouse embryos, due to the high contrast between the different tissue layers (Fig. 3). Other soft-tissue structures such as the central nervous system, peripheral nerves and blood vessels can also be imaged with high contrast (Fig. 4).
Virtual serial sections provide a reliable data source for analyzing the 3-D shape of embryonic structures. In fact, using histological serial sectioning followed by contouring and 3-D reconstruction is a long process requiring expert hands for good results (Viriot et al., 1997, 2000). Furthermore, this method has intrinsic sources of error when it comes to the alignment of the successive sections, despite efficient newly developed tools (Handschuh et al., 2010).
High-resolution microtomography has been previously used in the study of fossil embryos (Fernandez et al., 2012) and other mineralized soft-tissues (Pradel et al., 2009, Pradel, 2009) with a 5 μm voxel size. Smaller voxel sizes have been used in order to describe the fine structure of fossil vertebrate bone at 0.678 μm (Sanchez et al., 2012) and enamel at 0.695 μm (Tafforeau et al., 2012).
Furthermore, attempts have previously been made for visualizing soft-tissues using synchrotron light (Rao et al., 2010; Cooper et al., 2011). A recent study used in vivo phase contrast microtomography in order to describe cell motion during Xenopus gastrulation (Moosmann et al., 2013). Raj et al. (2014) have shown that synchrotron light with a silver-based contrast agent provides satisfactory images of early mouse embryos with a resolution of 4–10 μm.
By combining iodine staining (Metscher, 2009) and specific settings of the synchrotron beamline, we were able to perform rapid scans on full embryos at stages from E11.5 to E13.5 and on embryo heads from stages from E14.5 to E17.5, at an optimal voxel size of 0.695 μm. The technique we report can be used to study the developmental dynamics and growth morphology of rarely described structures such as Rathke's pouch, with an image quality comparable to histology (Khonsari et al., 2013). Rapid scans of full embryos can also be used for the screening of developmental anomalies in transgenic strains, with the possibility of obtaining full head sections and performing straightforward 3-D reconstructions of the relevant structures. This technique is thus of use both for the screening of anomalies in embryos and for the detailed 2-D and 3-D anatomical study of growing soft-tissue structures.
R.H. Khonsari, C. Healy, A. Ohazama, P.T. Sharpe
Department of Craniofacial Development and Stem Cell Biology Dental Institute King's College London London, UK
AP-HP, Hôpital Pitié-Salpêtrière Service de Chirurgie Maxillofaciale et Stomatologie Paris, F-75013 France
UPMC Université Paris 06 F-75005 Paris, France
UMR CNRS-MNHN-UPMC 7207 & UMR CNRS-MNHN 7179 Muséum national d'histoire naturelle Paris, France
Laboratory for Evolutionary Morphology RIKEN Center for Developmental Biology Kobe, Japan
C. Charles and L. Viriot
Evo-Devo of Vertebrate Dentition Institut de Génomique Fonctionnelle de Lyon Université de Lyon, CNRS UMR 5242 UCBL 1, Ecole Normale Supérieure de Lyon Lyon, France
European Synchrotron Radiation Facility Grenoble, France