Micro-computed tomography-based phenotypic approaches in embryology: procedural artifacts on assessments of embryonic craniofacial growth and development

Abstract

BackgroundGrowing demand for three dimensional (3D) digital images of embryos for purposes of phenotypic assessment drives implementation of new histological and imaging techniques. Among these micro-computed tomography (μCT) has recently been utilized as an effective and practical method for generating images at resolutions permitting 3D quantitative analysis of gross morphological attributes of developing tissues and organs in embryonic mice. However, histological processing in preparation for μCT scanning induces changes in organ size and shape. Establishing normative expectations for experimentally induced changes in size and shape will be an important feature of 3D μCT-based phenotypic assessments, especially if quantifying differences in the values of those parameters between comparison sets of developing embryos is a primary aim. Toward that end, we assessed the nature and degree of morphological artifacts attending μCT scanning following use of common fixatives, using a two dimensional (2D) landmark geometric morphometric approach to track the accumulation of distortions affecting the embryonic head from the native, uterine state through to fixation and subsequent scanning.ResultsBouin's fixation reduced average centroid sizes of embryonic mouse crania by approximately 30% and substantially altered the morphometric shape, as measured by the shift in Procrustes distance, from the unfixed state, after the data were normalized for naturally occurring shape variation. Subsequent μCT scanning produced negligible changes in size but did appear to reduce or even reverse fixation-induced random shape changes. Mixtures of paraformaldehyde + glutaraldehyde reduced average centroid sizes by 2-3%. Changes in craniofacial shape progressively increased post-fixation.ConclusionsThe degree to which artifacts are introduced in the generation of random craniofacial shape variation relates to the degree of specimen dehydration during the initial fixation. Fixation methods that better maintain original craniofacial dimensions at reduced levels of dehydration and tissue shrinkage lead to the progressive accumulation of random shape variation during handling and data acquisition. In general, to the degree that embryonic organ size and shape factor into μCT-based phenotypic assessments, procedurally induced artifacts associated with fixation and scanning will influence results. Experimental designs will need to address these significant effects, either by employing alternative methods that minimize artifacts in the region of focus or in the interpretation of statistical patterns.

Analyzing Fluctuating Asymmetry with Geometric Morphometrics: Concepts, Methods, and Applications

“…Even freezing can affect the shape of fish significantly [308]. Naturally, such artifacts from preservation are most serious for soft tissues such as embryos [309] or brain tissue [310]. Similarly, very small specimens such as mites [128] or the wings of parasitoid wasps [129] need to be mounted on microscope slides, which may produce artifacts due to the positioning of specimens on the slide or distortions due to the pressure from the coverslip.…”

“…It may be possible to make measurements before and after preservation [305,[307][308][309][310], which makes it possible to quantify any artifacts and to their magnitude in relation to the biological effects under study. An even better option is to avoid preservation altogether.…”

X-Ray Microtomographic Imaging of Intact Vertebrate Embryos

“…• 4F1G or another formaldehyde-glutaraldehyde fixative (as used for electron microscopy) 4F1G (McDowell and Trump 1976) results in less shrinkage of embryonic tissues than some other fixatives (Metscher 2009b;Schmidt et al 2010). …”

Genes into geometry: imaging for mouse development in 3D

“…More complex geometries are impossible to visualize in anything but 3D space. Facial development, in which the relatively simple branchial arches develop into the various structures of the face, throat and neck, requires 3D interpretation as reported with micro-CT [28][29][30] and with OPT [31]. Patterning of the limb is similarly complex and requires the coordinated development of tendons, muscles, bones and vasculature and is well shown with OPT [32][33][34].…”