使用共聚焦显微镜对连续物理切片的大型组织标本进行体积重建,并通过弹性配准校正切割变形。
Volume reconstruction of large tissue specimens from serial physical sections using confocal microscopy and correction of cutting deformations by elastic registration.
作者信息
Capek Martin, Brůza Petr, Janácek Jirí, Karen Petr, Kubínová Lucie, Vagnerová Radomíra
机构信息
Department of Biomathematics, Institute of Physiology, Academy of Sciences of the Czech Republic, v.v.i., Vídenská 1083, 142 20 Prague 4 - Krc, Czech Republic.
出版信息
Microsc Res Tech. 2009 Feb;72(2):110-9. doi: 10.1002/jemt.20652.
A set of methods leading to volume reconstruction of biological specimens larger than the field of view of a confocal laser scanning microscope (CLSM) is presented. Large tissue specimens are cut into thin physical slices and volume data sets are captured from all studied physical slices by CLSM. Overlapping spatial tiles of the same physical slice are stitched in horizontal direction. Image volumes of successive physical slices are linked in axial direction by applying an elastic registration algorithm to compensate for deformations because of cutting the specimen. We present a method enabling us to keep true object morphology using a priori information about the shape and size of the specimen, available from images of the cutting planes captured by a USB light microscope immediately before cutting the specimen by a microtome. The errors introduced by elastic registration are evaluated using a stereological point counting method and the Procrustes distance. Finally, the images are enhanced to compensate for the effect of the light attenuation with depth and visualized by a hardware accelerated volume rendering. Algorithmic steps of the reconstruction, namely elastic registration, object morphology preservation, image enhancement, and volume visualization, are implemented in a new Rapid3D software package. Because confocal microscopes get more and more frequently used in scientific laboratories, the described volume reconstruction may become an easy-to-apply tool to study large biological objects, tissues, and organs in histology, embryology, evolution biology, and developmental biology. In this work, we demonstrate the reconstruction using a postcranial part of a 17-day-old laboratory Wistar rat embryo.
本文介绍了一套用于对大于共聚焦激光扫描显微镜(CLSM)视野的生物标本进行体积重建的方法。将大型组织标本切成薄的物理切片,然后通过CLSM从所有研究的物理切片中获取体积数据集。对同一物理切片的重叠空间切片在水平方向上进行拼接。通过应用弹性配准算法来补偿由于切割标本而产生的变形,从而在轴向方向上连接连续物理切片的图像体积。我们提出了一种方法,利用从切片前立即用USB光学显微镜拍摄的切割平面图像中获得的关于标本形状和大小的先验信息,使我们能够保持真实的物体形态。使用体视学点计数法和普氏距离评估弹性配准引入的误差。最后,对图像进行增强以补偿光随深度衰减的影响,并通过硬件加速体绘制进行可视化。重建的算法步骤,即弹性配准、物体形态保留、图像增强和体积可视化,在一个新的Rapid3D软件包中实现。由于共聚焦显微镜在科学实验室中越来越频繁地使用,所描述的体积重建可能会成为研究组织学、胚胎学、进化生物学和发育生物学中的大型生物物体、组织和器官的一种易于应用的工具。在这项工作中,我们展示了使用17天大的实验室Wistar大鼠胚胎的颅后部分进行的重建。