Becker R L, Mikel U V, Oliver W R, Sesterhenn I A
Department of Cellular Pathology, Armed Forces Institute of Pathology, Washington, DC 20306-6000, USA.
Anal Quant Cytol Histol. 1996 Oct;18(5):405-9.
To compare two visual enumeration methods to determine whether the confocal approach yielded better counts of chromosome-specific hybridization sites.
Brightfield microscopy was used to count in situ hybridization (ISH) sites in 4-microns tissue sections. Confocal microscopy was used to collect three-dimensional (3D) data sets from fluorescence in situ hybridization (FISH) preparations made with sections of various thicknesses. Analysis of the confocal images relied on custom-built interactive visualization software.
The confocal method yielded higher average counts of hybridization sites per nucleus due to fewer truncated nuclei in thicker sections and to visual exclusion of the truncated nuclei that remained. Optimal section thickness was 8-12 microns. Limited penetration by FISH reagents restricted the use of thicker sections.
Analysis of intact nuclei visualized in three dimensions was more sensitive in demonstrating high centromere number than was brightfield ISH analysis of 4-microns sections. Improvements in semiautomated interactive software may make the confocal approach practical for accurate evaluation of chromosome number in precise histologic contexts.
比较两种视觉计数方法，以确定共聚焦方法是否能产生更好的染色体特异性杂交位点计数。
使用明场显微镜对4微米厚的组织切片中的原位杂交（ISH）位点进行计数。使用共聚焦显微镜从用不同厚度切片制作的荧光原位杂交（FISH）制剂中收集三维（3D）数据集。共聚焦图像的分析依赖于定制的交互式可视化软件。
由于较厚切片中截断核较少，且对剩余的截断核进行了视觉排除，共聚焦方法产生的每个细胞核杂交位点平均计数更高。最佳切片厚度为8 - 12微米。FISH试剂的穿透有限限制了较厚切片的使用。
与对4微米切片进行明场ISH分析相比，对三维可视化的完整细胞核进行分析在显示高着丝粒数方面更敏感。半自动交互式软件的改进可能使共聚焦方法在精确的组织学背景下准确评估染色体数目方面变得实用。
