An optimized, fully automated system for fast and accurate identification of chromosomal rearrangements by multiplex-FISH (M-FISH).
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.
Eils
R
R
Interdisciplinary Center of Scientific Computing, University of Heidelberg, Heidelberg (Germany).
Uhrig
S
S
Saracoglu
K
K
Sätzler
K
K
Bolzer
A
A
Petersen
I
I
Chassery
J
J
Ganser
M
M
Speicher
M R
MR
D002869:Chromosome Aberrations; D002871:Chromosome Banding / Q000379:methods; D004273:DNA, Neoplasm / Q000032:analysis*; D005456:Fluorescent Dyes; D015321:Gene Rearrangement; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007211:Indoles; D008856:Microscopy, Fluorescence / Q000379:methods
D004273:DNA, Neoplasm; D005456:Fluorescent Dyes; D007211:Indoles; C007293:DAPI