Multicolor Imaging

Multicolor FISH Analysis with Isis

The software upgrade for multicolor FISH (mFISH) and multicolor chromosome banding (mBAND).

Multicolor FISH assays are used for a precise assessment of complex chromosomal rearrangements. The assays are based on combinations of fluorochromes which are interpreted by the analysis software. The method of combinatorial labeling effectively multiplies the number of usable labels which would otherwise be limited to the 7 or 8 distinguishable individual fluorochromes.

Multicolor FISH

Multicolor-FISH (mFISH) is a method to facilitate analysis of each single chromosome or chromosome part of a metaphase. Thus, marker chromosomes, complex chromosomal rearrangements, and all numerical aberrations can be visualized simultaneously in a single hybridization experiment. By analyzing the color ratios of all pixels, the MetaSystems mFISH/mBAND software unambiguously assigns any region of the image to the respective chromosome class. The filter based mFISH approach facilitates a direct inspection of the original hybridization signals and, thus, overcomes the need for additional control experiments with conventional chromosomal paints.

MetaSystems provides an integrated system-plus-reagents solution for mFISH. The 24XCyte mFISH probe kit consists of a set of chromosome painting probes with specific fluorochrome combinations covering the human genome. mFISH kits are also available for mouse, rat, and Chinese hamster ovary (CHO) chromosomes (21XMouse, 22XRat, 12XCHamster).

Versatile tools for precise chromosome separation and for rapid chromosome classification help speeding up the workflow. Chromosome classification is either done based on DAPI banding or by using the fluorochrome combinations. mFISH chromosomes can be displayed in three different pseudo color display modes in order to facilitate the analysis: maximum saturation (solid color), DAPI image overlaid with the pseudo color and standard pseudo color image.

Analysis of mFISH images in Isis is easy even if complex aberrations are involved. The Pixel Info / Extended Pixel Info tools display the chromosome class assigned to the selected pixel. The reliability index of the assignment can also be displayed. The False Color Binary Display virtually transforms the mFISH image into a series of hybridizations with 3 individual chromosome paints. This tool was introduced to facilitate the analysis of mFISH images with numerous chromosome rearrangements that cannot be identified "at a glance". Ambiguous color signatures can optionally be excluded from the classification. This way, regions with insufficient hybridization are easily identified and suppressed. The Single Color Gallery is a separate window in which all five color channels are displayed for a selected chromosome. Additionally the false color image, an inverted DAPI image, and the fluorochrome profiles are shown. With the help of the Single Color Gallery the identification of rearrangements is extremely easy and straightforward.

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Multicolor Banding

Multicolor chromosome banding (mBAND), a proprietary technique of MetaSystems, principally uses the technique of multicolor FISH (mFISH) and applies it to the detection of intra-chromosomal rearrangements. In detail, the probes (XCyte mBand probes) labeled with different proportions of two or more fluorochromes are hybridized to a single chromosome pair. Each probe consists of overlapping partial chromosome paints. The changing fluorescence intensity ratios along the chromosomes are used by the Isis software to assign different pseudo-colors to specific chromosome regions. The result is a reproducible high-resolution false-color banding pattern allowing for identification and localization of intra-chromosomal aberrations.

The resolution of the mBAND pattern can be modulated in order to get the most suitable image for analysis. A resolution equivalent to 550-band level for G-bands can be easily achieved. mBAND patterns are independent of chromatin condensation. Though mBAND bands resemble an artificial banding pattern, they can be directly correlated with ISCN bands using the single color gallery of Isis. Hence, precise localization of breakpoints is possible.

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