An optimized, fully automated system for fast and accurate identification of chromosomal rearrangements by multiplex-FISH (M-FISH)

Cytogenet Cell Genet. 1998;82(3-4):160-71. doi: 10.1159/000015092.


Multiplex-FISH (M-FISH) is a recently developed technique by which each of the two dozen human chromosomes-the 22 autosomes and the X and Y sex chromosomes-can be stained or "painted" with uniquely distinctive colors. Using a combinatorial labeling technique and a specially designed filter set, each DNA probe can be identified by its unique spectral signature. Here we present several significant optimizations of the M-FISH technology. First, a new strategy for labeling the probes is described which allows for easy and fast production of the complex M-FISH probe mix. Second, a newly developed, completely motorized microscope equipped with an eight-position filter wheel and a new generation of filter sets is presented that allows fully automatic imaging of a complete metaphase spread within seconds. Third, to determine the characteristic spectral signatures for all different combinations of fluorochromes, we developed a novel multichannel image analysis method. The spectral analysis is solely guided by the image information itself and does not require any user interaction. A complete analysis of a metaphase spread can be accomplished in less than 3 min. Sophisticated built-in quality controls were developed, and the value of visual inspection of M-FISH images as a simple means of controlling the computer-generated chromosome classification are illustrated. In addition, we discuss advantages of adding new fluorochromes to the traditionally used five fluorochromes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromosome Aberrations*
  • Chromosome Banding / methods
  • DNA, Neoplasm / analysis*
  • Fluorescent Dyes
  • Gene Rearrangement*
  • Humans
  • Image Processing, Computer-Assisted / methods
  • In Situ Hybridization, Fluorescence / methods*
  • Indoles
  • Microscopy, Fluorescence / methods


  • DNA, Neoplasm
  • Fluorescent Dyes
  • Indoles
  • DAPI