Schmidt Eric J, Parsons Trish E, Jamniczky Heather A, Gitelman Julian, Trpkov Cvett, Boughner Julia C, Logan C Cairine, Sensen Christoph W, Hallgrímsson Benedikt
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.
BMC Dev Biol. 2010 Feb 17;10:18. doi: 10.1186/1471-213X-10-18.
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.
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.
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.
出于表型评估目的，对胚胎三维（3D）数字图像的需求不断增长，这推动了新的组织学和成像技术的应用。其中，微计算机断层扫描（microCT）最近已被用作一种有效且实用的方法，以生成分辨率足以对胚胎小鼠发育中的组织和器官的大体形态特征进行3D定量分析的图像。然而，为进行microCT扫描而进行的组织学处理会引起器官大小和形状的变化。为实验诱导的大小和形状变化建立规范预期将是基于3D microCT的表型评估的一个重要特征，特别是如果量化发育胚胎比较组之间这些参数值的差异是主要目标的话。为此，我们使用二维（2D）地标几何形态测量方法，评估了使用常见固定剂后microCT扫描产生的形态学伪影的性质和程度，以追踪从天然子宫状态到固定及后续扫描过程中影响胚胎头部的变形累积情况。
经布安氏固定后，胚胎小鼠颅骨的平均质心大小减少了约30%，并且在对自然发生的形状变化进行数据归一化后，从未固定状态测量的形态计量形状发生了显著改变，这通过普氏距离的变化来衡量。随后的microCT扫描在大小方面产生的变化可忽略不计，但似乎确实减少甚至逆转了固定引起的随机形状变化。多聚甲醛 + 戊二醛混合物使平均质心大小减少了2 - 3%。固定后颅面形状的变化逐渐增加。
在随机颅面形状变化生成过程中引入伪影的程度与初始固定过程中样本脱水的程度有关。在较低脱水水平和组织收缩情况下能更好地保持原始颅面尺寸的固定方法，会导致在处理和数据采集过程中随机形状变化的逐渐累积。一般来说，就胚胎器官大小和形状对基于microCT的表型评估的影响程度而言，与固定和扫描相关的程序诱导伪影将影响结果。实验设计需要通过采用在焦点区域最小化伪影的替代方法或在统计模式解释中解决这些显著影响。
