Multicolor FISH
TOPICS: |1| 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23
FIGURES: | 1 | 2 | 3 | 4 | 5 | 6 |7 | 8 | 9 | 10 | 11 |TABLES: | 1 | 2 | 3 | 4 | 5 | 6 | 7

Combinatorial labeling.
One main advantage of FISH over radioactive in situ hybridization is the capability of hybridizing and differentiating more than one DNA probe at the same time on nuclei and chromosomes. This allows mapping probes relative to one another in interphase or metaphase, quantitative analyses as in CGH, or simultaneous identification of all human chromosomes as in M-FISH.

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
If the fluorescent microscope is equipped with filters allowing excitation and detection of three or more fluorescent colors, three probes can be simultaneously hybridized and detected, providing distinctive advantages over single or double probe hybridizations. For example, this allows visualization of the centromere of a chromosome (diagnosis of aneuploidy) in one color, along with the centromeres of the X and Y chromosomes in two different colors, to provide simultaneous sex diagnosis (Fig. 10a). This triple color strategy allows rapid and efficient interphase (Fig. 10b) and metaphase (Fig. 10c) mapping of DNA probes relative to one another. Use of three different fluors is superior to combinatorial labeling of three probes with two fluors, as it resolves unequivocally very close or partially overlapped signals.

Multiple color FISH
(please check the M-FISH schemes and the CM-FISH, TM-FISH and CCK pages)

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.