Silver-Albumin Tissue Staining Protocol to Visualize Odontogenesis in Whole Embryos
Visualizing tooth organs from their earliest inception as they actually appear in three dimensions has, until recently, been difficult due to the technical obstacle of imaging these tiny, translucent, low-density embryonic craniodental tissues. Related to this obstacle, quantifying craniodental morphology has been confounded by the time consuming need to physically section and then digitally photograph and reconstruct these images of tissues into 3D volumes. Here we provide a simple solution in the form of an overnight silver albumin tissue stain for whole embryos. Because it is differentially absorbed by embryonic tissues, this stain generates the contrast needed to detect and visualize unmineralized dental tissues. Stained specimens can be scanned using either desktop or synchrotron micro-computed tomography systems, generating digital 3D datasets of whole embryos that can immediately be used to assess dental morphology and histology. Craniodental structures can then be measured with high precision and accuracy using 3D image analysis software.
Key wordsContrast agent Tooth development Micro-computed tomography 3D imaging Morphogenesis Virtual histology Synchrotron
We are grateful to Karen Yuen and the College of Medicine Histology Core Facility for the help and resources required to optimize this staining technique. We also thank College of Medicine Vivarium staff, particularly Carmen Whitehead for maintaining JCB’s mouse colony. JCB and DMLC are each funded by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC; JCB (#2011-402148, #2016-05177); DMLC (#2014-05563) and by the Canadian Foundation for Innovation (CFI infrastructure grants to JCB (#29037). This work was also supported by the Canadian Institutes of Health Research (CIHR)-THRUST Fellowship program. The method described in this article was developed in part at the Canadian Light Source, which is supported by NSERC, CIHR, the National Research Council Canada, Province of Saskatchewan, Western Economic Diversification Canada, and University of Saskatchewan.
- 1.Nanci A (2012) Ten cate’s oral histology: development, structure and function, 8th edn. Elsevier, TorontoGoogle Scholar
- 2.Berkovitz BKB, Holland GR, Moxham BJ (2017) Oral anatomy, histology and embryology, 5th edn. Elsevier, TorontoGoogle Scholar
- 13.Gignac PM, Kley NJ, Clarke JA, Colbert MW, Morhardt AC, Cerio D, Cost IN, Cox PG, Daza JD, Early CM et al (2016) Diffusible iodine-based contrast-enhanced computed tomography (diceCT): an emerging tool for rapid, high-resolution, 3-D imaging of metazoan soft tissues. J Anat 228:889–909CrossRefGoogle Scholar
- 14.Schmidt EJ, Parsons TE, Jamniczky HA, Gitelman J, Trpkov C, Boughner JC, Logan CC, Sensen CW, Hallgrimsson B (2010) Micro-computed tomography-based phenotypic approaches in embryology: procedural artifacts on assessments of embryonic craniofacial growth and development. BMC Dev Biol 10:18CrossRefGoogle Scholar