Isis mFISH Multicolor Chromosome Analysis

General

MetaSystems provides an integrated system-plus-reagents (24 XCyte mFISH kit) solution for multicolor chromosome analysis. mFISH analysis is based on a DNA probe kit comprising whole chromosome painting probes labelled with different fluorochromes or fluorochrome combinations. The mFISH software analyzes the color information and identifies the chromosomal origin of each individual pixel within the image. Even complex rearrangements are readily detectable and fully documented.

Isis mFISH Options and Links
MetaSystems also provides the 24 XCyte mFISH DNA Probe Kits.	more...
mFISH is a module for the FISH imaging software Isis.	more...
The mFISH module allows for the analysis of multi-color banding (mBAND)	more...

Image acquisition

Image acquisition is performed using a standard fluorescence microscope equipped with highly selective fluorescence filters. The different fluorochromes are captured into six (up to nine) individual color channels. Motorized filter change is supported. Integration times are automatically controlled by the software. The short total acquisition time of typically 20 seconds under narrow band illumination avoids photo bleaching of the specimen.

In combination with the revolutionary Metafer AutoCapt module for the automated metaphase finder Metafer MSearch, the image acquisition can be done fully automatically. This configuration allows to detect the metaphases on the slide (in the counterstain mode), and subsequently let AutoCapt acquire the images into Isis compatible file formats. Since the positions of the metaphases on the slides are stored with the images, it is also very easy to relocate any cell and to revise it under the microscope.

After automatic pre-processing including automatic background correction, thresholding the counterstain image and interactive or automatic registration of the color channels the result can be visualized as either RGB or false color image. Trainable color classifiers provide highest flexibility and specificity.

Pseudo color assignment

Pseudo color assignment is used to display the chromosomes in distinguishable pseudocolors. The software performs a color classification to determine the chromosomal origin of each pixel within the image by its color signature, and assigns a pre-selected pseudo-color to all pixels corresponding to a particular chromosome. Inter-chromosomal rearrangements will show up as color changes within the aberrant chromosome.

Isis adapts automatically to a new labelling scheme if classifier training is performed. The program "learns" the color composition of each chromosome and assigns suitable pseudo colors to the chromosomes for optimum discrimination. This approach works for combinatorial as well as for ratio labelling.

mFISH analysis

Visually comparing pseudo colors may not be sufficient to identify chromosome fragments in complex translocations. Sophisticated display features facilitate rearrangement analysis:

The "Point-and-Identify" feature opens a so called "tool tip" window showing the chromosome class, if the mouse pointer is moved over a chromosome. With this tool chromosomes can be identified precisely without the need to compare the pseudocolors directly.

Three different pseudo color modes (standard, saturated and counterstain modulated) ease the precise analysis of any metaphase. If the pseudocolors should be seen very brightly, the staurated (100%) mode is the display of choice. However, details of the counterstain banding pattern are not visible in this mode. The counterstain modulated view combines the counterstain banding pattern with pseudo-colors.

The "Binary Display" function provides differential translocation highlighting by displaying up to 3 selected chromosomes in red, green, and blue, while showing all remaining chromosomes without color. The list of chromosomes allows for scrolling through the whole metaphase and so easily identifying any rearrangement.

Another powerful tool to do the final diagnosis is the "Single Color Gallery". Clicking on a chromosome, a small window is opened, which shows the false color image, each fluorochrome channel and the inverted counterstain image separately. Additionally, the color profile of chromosome and the false color scheme is displayed. An ideogram of the chromosome accompanies the counterstain image. Since each chromosome class has a unique hybridization pattern, any disruption due to rearrangements can be easily identified and even localized in the "Single Colour Gallery". Images of the "Single Colour Gallery" may be exported or printed to document the diagnosis.