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

Schmidt EJ, Parsons TE, Jamniczky HA, Gitelman J, Trpkov C, Boughner JC, Logan CC, Sensen CW, Hallgrímsson B - BMC Dev. Biol. (2010)

Bottom Line: Subsequent microCT 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%.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology & Anatomy, The McCaig Bone and Joint Institute, and the Alberta Children's Hospital Institute for Child and Maternal Health, University of Calgary, Calgary, AB, Canada.

ABSTRACT

Background: Growing 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 (microCT) 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 microCT 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 microCT-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 microCT 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.

Results: Bouin'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 microCT 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.

Conclusions: The 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 microCT-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.

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A, PCA results for the raw Procrustes transformed data by fixation method and treatment step. Wireframes as in Figure 1A. B, Regressions of shape on craniofacial (centroid) size showing variable effects of treatments within experimental groups (experimental groups left to right: Bouin's, 1% glutaraldehyde, 5% glutaraldehyde).
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Figure 4: A, PCA results for the raw Procrustes transformed data by fixation method and treatment step. Wireframes as in Figure 1A. B, Regressions of shape on craniofacial (centroid) size showing variable effects of treatments within experimental groups (experimental groups left to right: Bouin's, 1% glutaraldehyde, 5% glutaraldehyde).

Mentions: PCA results for the raw Procrustes transformed data by fixation method and treatment steps are shown in Figure 4A. Wireframe distortions along PC1 and PC2 indicate the presence of coherent, systematic variation within the raw dataset. Regressions of shape on craniofacial (centroid) size show variable effects of treatments within experimental groups (Figure 4B). The positive slopes of the regression lines for each of the three experimental groups demonstrate an ontogenetic component of variation in which shape change scales with size, a result not unexpected. Interestingly, unlike in either glutaraldehyde experimental group, there is within the Bouin's experimental group a conspicuous shift in y-intercept values between regression lines representing scaling effects associated with harvesting and with fixation and μCT scanning. This possibly indicates that in addition to any naturally occurring allometric variation, Bouin's fixation dramatically adds an additional source of variation. Since slopes representing the effects of fixation and post-scanning are shifted leftward, substantial craniofacial shrinkage associated with fixation is likely occurring. The trends within either glutaraldehyde experimental group, though expected, are not as apparent.


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

Schmidt EJ, Parsons TE, Jamniczky HA, Gitelman J, Trpkov C, Boughner JC, Logan CC, Sensen CW, Hallgrímsson B - BMC Dev. Biol. (2010)

A, PCA results for the raw Procrustes transformed data by fixation method and treatment step. Wireframes as in Figure 1A. B, Regressions of shape on craniofacial (centroid) size showing variable effects of treatments within experimental groups (experimental groups left to right: Bouin's, 1% glutaraldehyde, 5% glutaraldehyde).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2836989&req=5

Figure 4: A, PCA results for the raw Procrustes transformed data by fixation method and treatment step. Wireframes as in Figure 1A. B, Regressions of shape on craniofacial (centroid) size showing variable effects of treatments within experimental groups (experimental groups left to right: Bouin's, 1% glutaraldehyde, 5% glutaraldehyde).
Mentions: PCA results for the raw Procrustes transformed data by fixation method and treatment steps are shown in Figure 4A. Wireframe distortions along PC1 and PC2 indicate the presence of coherent, systematic variation within the raw dataset. Regressions of shape on craniofacial (centroid) size show variable effects of treatments within experimental groups (Figure 4B). The positive slopes of the regression lines for each of the three experimental groups demonstrate an ontogenetic component of variation in which shape change scales with size, a result not unexpected. Interestingly, unlike in either glutaraldehyde experimental group, there is within the Bouin's experimental group a conspicuous shift in y-intercept values between regression lines representing scaling effects associated with harvesting and with fixation and μCT scanning. This possibly indicates that in addition to any naturally occurring allometric variation, Bouin's fixation dramatically adds an additional source of variation. Since slopes representing the effects of fixation and post-scanning are shifted leftward, substantial craniofacial shrinkage associated with fixation is likely occurring. The trends within either glutaraldehyde experimental group, though expected, are not as apparent.

Bottom Line: Subsequent microCT 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%.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology & Anatomy, The McCaig Bone and Joint Institute, and the Alberta Children's Hospital Institute for Child and Maternal Health, University of Calgary, Calgary, AB, Canada.

ABSTRACT

Background: Growing 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 (microCT) 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 microCT 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 microCT-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 microCT 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.

Results: Bouin'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 microCT 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.

Conclusions: The 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 microCT-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.

Show MeSH
Related in: MedlinePlus