Walter J, Joffe B, Bolzer A, Albiez H, Benedetti P A, Müller S, Speicher M R, Cremer T, Cremer M, Solovei I
Chair of Anthropology and Human Genetics, Department of Biology II, Ludwig-Maximilians-University, Martinsried, Germany.
Cytogenet Genome Res. 2006;114(3-4):367-78. doi: 10.1159/000094227.
The article reviews the existing methods of multicolor FISH on nuclear targets, first of all, interphase chromosomes. FISH proper and image acquisition are considered as two related components of a single process. We discuss (1) M-FISH (combinatorial labeling + deconvolution + wide-field microscopy); (2) multicolor labeling + SIM (structured illumination microscopy); (3) the standard approach to multicolor FISH + CLSM (confocal laser scanning microscopy; one fluorochrome - one color channel); (4) combinatorial labeling + CLSM; (5) non-combinatorial labeling + CLSM + linear unmixing. Two related issues, deconvolution of images acquired with CLSM and correction of data for chromatic Z-shift, are also discussed. All methods are illustrated with practical examples. Finally, several rules of thumb helping to choose an optimal labeling + microscopy combination for the planned experiment are suggested.
本文回顾了针对细胞核靶点，首先是间期染色体的多色荧光原位杂交（FISH）的现有方法。FISH本身和图像采集被视为一个单一过程的两个相关组成部分。我们讨论了：（1）多色FISH（组合标记+反卷积+宽场显微镜）；（2）多色标记+结构光照显微镜（SIM）；（3）多色FISH+共聚焦激光扫描显微镜（CLSM）的标准方法（一种荧光染料 - 一个颜色通道）；（4）组合标记+CLSM；（5）非组合标记+CLSM+线性解混。还讨论了两个相关问题，即CLSM采集图像的反卷积和色移数据校正。所有方法均配有实际示例说明。最后，提出了几条经验法则，有助于为计划中的实验选择最佳的标记+显微镜组合。
