Two fluorophores can be used to label and detect three different DNA probes by combinatorial labeling. (A more thorough description of the concept can be found here). For example, if one probe is labeled with FITC-dUTP (green), and another probe with rhodamine-dUPT (red), a third probe can be labeled with both FITC-dUPT and rhodamine-dUTP (green and red). When examined under the fluorescent microscope, the combination of red and green will appear yellow (Fig. 9l), allowing simultaneous differentiation of three probes using only two dyes. Similarly, three basic colors provide seven possible combinations, and so on, based on a 2^n-1 formula.
Triple color FISH
In M-FISH, chromosomes are hybridized with whole chromosome painting probes, labeled by combinations of five different fluorophores (scheme here). After hybridization, gray-scale images of each fluorophore are captured with a digital camera, and the five images are pseudocolored by the computer. The combinatorial labeling algorithm used allows separation and identification of all chromosomes by their color. This process is simply depicted for five different chromosome pairs in the figure below. Arrow show cross-talk between the channels: when filters are not properly manufactured or chosen, a fluorophore can be excited and visualized through an adjacent filter (FITC is partially visulized through the Cy3 filter or Cy3.5 is visulized through the Cy5 filter). These situations should be corrected either by requesting appropriate filters from the manufacturers or by changing the fluorophores used.
An example of a normal M-FISH analysis of a human metaphase is shown in the figure below.
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Last modified on: Feb12, 2001