关于三维保存的间期细胞核荧光原位杂交中的多种颜色

Towards many colors in FISH on 3D-preserved interphase nuclei.

作者信息

Walter J, Joffe B, Bolzer A, Albiez H, Benedetti P A, Müller S, Speicher M R, Cremer T, Cremer M, Solovei I

机构信息

Chair of Anthropology and Human Genetics, Department of Biology II, Ludwig-Maximilians-University, Martinsried, Germany.

出版信息

Cytogenet Genome Res. 2006;114(3-4):367-78. doi: 10.1159/000094227.

DOI:10.1159/000094227

PMID:16954680

Abstract

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.

摘要

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。