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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-D, photographic series of a single embryo from the 5% glutaraldehyde experimental group representing general workflow repeated for all individuals in sample. A, freshly harvested, unfixed specimen representing initial photographs overlain with landmarking scheme: red fan samples midbrain, green fan samples left telencephalon. Wireframe outline of landmarks in blue. B, same embryo fixed with 4% formaldehyde + 5% glutaraldehyde. C, 3D μCT scan. D, final digital photograph of series taken after μCT scanning. E-G, imaging series of an individual representing workflow for individuals from Bouin's experimental group. E, Bouin's fixation. F, μCT scan. G, final photo of same specimen post μCT scan. Photographic series representing individuals of 4% formaldehyde + 1% glutaraldehyde experimental group not shown. A, B, D, E, and G at same magnification. H5%, harvested embryo of 5% glutaraldehyde experimental group. F5%, fixed embryo of 5% glutaraldehyde experimental group. PS5%, post-scanned embryo of 5% glutaraldehyde experimental group. FB, fixed embryo of Bouin's experimental group. PSB, post-scanned embryo of Bouin's experimental group. All embryos are shown in left lateral view.
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Figure 10: A-D, photographic series of a single embryo from the 5% glutaraldehyde experimental group representing general workflow repeated for all individuals in sample. A, freshly harvested, unfixed specimen representing initial photographs overlain with landmarking scheme: red fan samples midbrain, green fan samples left telencephalon. Wireframe outline of landmarks in blue. B, same embryo fixed with 4% formaldehyde + 5% glutaraldehyde. C, 3D μCT scan. D, final digital photograph of series taken after μCT scanning. E-G, imaging series of an individual representing workflow for individuals from Bouin's experimental group. E, Bouin's fixation. F, μCT scan. G, final photo of same specimen post μCT scan. Photographic series representing individuals of 4% formaldehyde + 1% glutaraldehyde experimental group not shown. A, B, D, E, and G at same magnification. H5%, harvested embryo of 5% glutaraldehyde experimental group. F5%, fixed embryo of 5% glutaraldehyde experimental group. PS5%, post-scanned embryo of 5% glutaraldehyde experimental group. FB, fixed embryo of Bouin's experimental group. PSB, post-scanned embryo of Bouin's experimental group. All embryos are shown in left lateral view.

Mentions: For each embryo, a series of digital photographs was taken documenting external gross morphology at each processing step: Initial embryo harvest, fixation, and after μCT scanning. Immediately following uterine extraction, the freshly harvested embryos were positioned on their right sides in a flat-bottomed dissecting dish filled with ice cold Liebovitz's L-15 Medium and photographed using a dissection microscope (32×) to capture left lateral views of the cranium (Figure 10A). Due to their irregular shapes, the embryos did not lie completely flat on their right side, but rather tended to be tilted so that the underlying right forebrain and nasal process were slightly visible beneath those of the left (Figures 10A-G). We chose to photograph the embryos in this resting position because it provided consistent positioning with minimal manipulation. We assume that while this positioning produces rotational error, that error is consistent. Subsequent photographs were taken of each embryo following fixation and again following μCT scanning (Figures 10D and 10G). For these latter photographs, the same standards were followed except that embryos were placed in a dissection dish filled with ice cold phosphate buffered saline (PBS). Following [33], we assume in this analysis that rotational error is present in our shape data and has two components. One should be random and therefore would not be expected to introduce any systematic biases. On the other hand, it is reasonable to expect that with fixation- and scan-induced changes to embryonic shape would come systematic effects on embryo positioning during photography. This component would be represented in the linear combinations comprising each principal component describing shape variation (Figures 3 and 7) but is not explicitly isolated and accounted for. It is considered to be a source of statistical nuisance that must be recognized as an inherent but difficult to avoid methodological shortcoming. Interpretations of our data should be qualified with respect to this deficiency.


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-D, photographic series of a single embryo from the 5% glutaraldehyde experimental group representing general workflow repeated for all individuals in sample. A, freshly harvested, unfixed specimen representing initial photographs overlain with landmarking scheme: red fan samples midbrain, green fan samples left telencephalon. Wireframe outline of landmarks in blue. B, same embryo fixed with 4% formaldehyde + 5% glutaraldehyde. C, 3D μCT scan. D, final digital photograph of series taken after μCT scanning. E-G, imaging series of an individual representing workflow for individuals from Bouin's experimental group. E, Bouin's fixation. F, μCT scan. G, final photo of same specimen post μCT scan. Photographic series representing individuals of 4% formaldehyde + 1% glutaraldehyde experimental group not shown. A, B, D, E, and G at same magnification. H5%, harvested embryo of 5% glutaraldehyde experimental group. F5%, fixed embryo of 5% glutaraldehyde experimental group. PS5%, post-scanned embryo of 5% glutaraldehyde experimental group. FB, fixed embryo of Bouin's experimental group. PSB, post-scanned embryo of Bouin's experimental group. All embryos are shown in left lateral view.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 10: A-D, photographic series of a single embryo from the 5% glutaraldehyde experimental group representing general workflow repeated for all individuals in sample. A, freshly harvested, unfixed specimen representing initial photographs overlain with landmarking scheme: red fan samples midbrain, green fan samples left telencephalon. Wireframe outline of landmarks in blue. B, same embryo fixed with 4% formaldehyde + 5% glutaraldehyde. C, 3D μCT scan. D, final digital photograph of series taken after μCT scanning. E-G, imaging series of an individual representing workflow for individuals from Bouin's experimental group. E, Bouin's fixation. F, μCT scan. G, final photo of same specimen post μCT scan. Photographic series representing individuals of 4% formaldehyde + 1% glutaraldehyde experimental group not shown. A, B, D, E, and G at same magnification. H5%, harvested embryo of 5% glutaraldehyde experimental group. F5%, fixed embryo of 5% glutaraldehyde experimental group. PS5%, post-scanned embryo of 5% glutaraldehyde experimental group. FB, fixed embryo of Bouin's experimental group. PSB, post-scanned embryo of Bouin's experimental group. All embryos are shown in left lateral view.
Mentions: For each embryo, a series of digital photographs was taken documenting external gross morphology at each processing step: Initial embryo harvest, fixation, and after μCT scanning. Immediately following uterine extraction, the freshly harvested embryos were positioned on their right sides in a flat-bottomed dissecting dish filled with ice cold Liebovitz's L-15 Medium and photographed using a dissection microscope (32×) to capture left lateral views of the cranium (Figure 10A). Due to their irregular shapes, the embryos did not lie completely flat on their right side, but rather tended to be tilted so that the underlying right forebrain and nasal process were slightly visible beneath those of the left (Figures 10A-G). We chose to photograph the embryos in this resting position because it provided consistent positioning with minimal manipulation. We assume that while this positioning produces rotational error, that error is consistent. Subsequent photographs were taken of each embryo following fixation and again following μCT scanning (Figures 10D and 10G). For these latter photographs, the same standards were followed except that embryos were placed in a dissection dish filled with ice cold phosphate buffered saline (PBS). Following [33], we assume in this analysis that rotational error is present in our shape data and has two components. One should be random and therefore would not be expected to introduce any systematic biases. On the other hand, it is reasonable to expect that with fixation- and scan-induced changes to embryonic shape would come systematic effects on embryo positioning during photography. This component would be represented in the linear combinations comprising each principal component describing shape variation (Figures 3 and 7) but is not explicitly isolated and accounted for. It is considered to be a source of statistical nuisance that must be recognized as an inherent but difficult to avoid methodological shortcoming. Interpretations of our data should be qualified with respect to this deficiency.

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