A simple setup for episcopic microtomy and a digital image processing workflow to acquire high-quality volume data and 3D surface models of small vertebrates
A simple setup for episcopic microtomy and a digital image processing workflow to acquire high-quality volume data and 3D surface models of small vertebrates
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DOI：
10.1007/s00435-017-0386-3
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发表时间：
2018-03-01
期刊：
影响因子：
1
通讯作者：
Haas, Alexander
中科院分区：
文献类型：
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作者：
Engelkes, Karolin;Friedrich, Frank;Haas, Alexander
体数据和数字三维(3D)表面模型在生物学中的使用迅速而稳定地增加。获取3D数据的方法有多种，其中包括电子显微镜成像技术。基于W eninger等人的块上染色镜检方法。(Anat Embryol197：341-348,1998)，我们描述了一种简单而通用的电镜式显微切开装置。它由一台消费型单反数码相机和标准的组织学设备组成。详细描述了块表面染色和成像、图像处理、堆叠对齐、表面生成(包括定制的AMIRA(A(R))宏)和3D模型编辑的工作流程。对于我们的样本标本(Alytes obstetricans；两栖动物：无尾两栖动物)，我们获得了像素大小为5.67x5.67A亩m(2)的图像。生成的图像堆栈允许区分不同的组织，并且非常适合创建3D表面模型。我们分析了不同样本和基准标记点的选择所达到的比对质量。基准点对对准质量有显著的正影响，最佳对准的最大平均对准误差约为44.7微米。我们进一步测试了微距镜头、延长管或遥控器组合使用的APS-C相机。微透镜和延长管的像素尺寸最小，为2.53×2.53微米(2)。考虑到数据质量和分辨率，并根据对象大小和研究目标，DSLR捕获的表观显微切割术可以替代其他技术，如传统的组织切片或微型计算机断层扫描。
The use of volume data and digital three-dimensional (3D) surface models in biology has increased quickly and steadily. Various methods are available to acquire 3D data, among them episcopic imaging techniques. Based on the episcopic microscopy with on-block staining protocol of Weninger et al. (Anat Embryol 197:341-348, 1998), we describe a simple and versatile setup for episcopic microtomy. It is composed of a consumer DSLR digital camera combined with standard histology equipment. The workflow of block surface staining and imaging, image processing, stack alignment, surface generation (including a custom Amira(A (R)) macro), and 3D model editing is described in detail. For our sample specimen (Alytes obstetricans; Amphibia: Anura) we obtained images with a pixel size of 5.67 x 5.67 A mu m(2). The generated image stacks allowed distinguishing different tissues and were well-suited for creating a 3D surface model. We analyzed the alignment quality achieved by various selections of specimen and fiducial marker spots. The fiducial spots had a significant positive effect on the alignment quality with the best alignment having a maximum mean alignment error of about 44.7 A mu m. We further tested the APS-C camera with combinations of macro lens, extension tube or teleconverter. The macro lens and extension tube yielded the smallest pixel size of 2.53 x 2.53 A mu m(2). Considering data quality and resolution, and depending on object sizes and research goals, DSLR captured episcopic microtomy can be an alternative to other techniques, such as traditional histological sectioning or micro-computed tomography.