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Ref: Nature Genetics 23 (3):263-4Get article in PDF format here
Color changing karyotyping (CCK):
an M-FISH/SKY alternative
1.CCK principle2.CCK examples3.CCK methods
2. Examples of CCK analysis
"3 + 3" algorithm
The "3+3" combination (Fig. 3a-g) mimics the use of six different "dyes" and is more robust, primarily because fewer chromosomes require simultaneous labeling with three different dyes/nucleotides (compare Table 1 with the scheme by Speicher et al., [Nature Genet 12, 368-375 (1996)]). Thus, the "3+3" approach should be preferred over the "3+2 or 2+3" combinations, which mimick the use of five "dyes". Two variations of the 3+3 protocol were tested (Table 2, #1 and #2). Because FITC, Cy3 and Cy5 are bright, stable and very separable fluorophores with available filters, combination #1 yielded the most robust fluorescent signals, with signal-to-noise (S/N) ratios from 1.8-4.5 for individual chromosomes. To obtain a DAPI image, however, this procedure required a fourth, DAPI filter. Combination #2 required the use of AMCA (aminomethyl-coumarin) in both the first and second image. As AMCA and DAPI spectra overlap, simultaneous DAPI counterstaining is not possible. DNA probes labeled with AMCA-conjugated nucleotides are more difficult to use, as AMCA photobleaches faster than other fluorophores tested. However, AMCA can be successfully used when conjugated to avidin or antibodies, a very useful feature for the 2+3 scheme.
Fig. 3.All images were processed in Adobe Photoshop. To allow simultaneous visualization of both labeled and unlabeled (faint) chromosomes and facilitate comparisons, captured images were only minimally processed, some background fluorescence being kept in every channel. As various painting probes had slightly different qualities, minimal processing kept these differences visible, closer to the real-life microscopic examination. a, b, c, depict the first, the second and the DAPI image of a normal metaphase using the 3+3 CCK algorithm #1 ( Table 1 and 2). In a, FITC was pseudocolored green, Cy3 red and Cy5 blue. In b, DNP was detected with FITC, DIG with Cy3 and BIO with Cy5 and pseudocoloring was identical to the "a" image. Chromosome numbers are shown in c (DAPI staining). d, e, depict the first and second images of a CCK analysis of the germ cell tumor line NT2D1, using the same 3+3 algorithm #1. Arrows in d indicate the chromosome translocations magnified in image f, in which the reverse DAPI staining is also shown.The colored dots and the corresponding numbers show the 3+3 color chart, which depicts the expected color of every chromosome pair in the first and second image, based on the colors assigned to each channel. For every chromosome, the first and second dots indicate the color of that chromosome pair in the first and second image, respectively. This charts can be used to visually "color-karyotype" all chromosomes. For example, the chart indicates that chromosome pairs 15 and 17 will be cyan (light blue) in the first image (a) but only 17 will change color (to red) in the second image (b), whereas 15 will lose its color.
"3 + 2" and "2 + 3" algorithms
The "2+3" or "3+2" algorithms mimic the use of five "dyes" (Fig. 1h-l), and chromosomes can be labeled according to the scheme suggested by Speicher et al. Several labeling combinations were compared (Table 2, #3 - #6), using a variety of three filter combinations. For general purposes, the most convenient combination is #4, the one using dyes which can be visualized through the three "standard" filters (DAPI, FITC and rhodamine/Texas Red). For this, AMCA labeled avidin was added to the detection system, together with the primary antibodies against DNP and DIG. "First image" included FITC, Texas Red and AMCA (avidin), whereas the second image included FITC (DNP), Texas Red (DIG) and DAPI. Alternatively, an AMCA-labeled IgG against DNP can be used at the first step, and biotin-FITC at the second step, to prevent any decrease of DNP signal due to short wavelength exposure. Although all "2+3" and "3+2" combinations performed equally well, another advantage of algorithm #4 was the use of common fluorescent dyes (fluorescein, rhodamine, coumarin derivatives), cheaper than cyanine dyes.
Fig. 4.The colored dots and the corresponding numbers show the 3+2 color chart, which depicts the expected color of every chromosome pair in the first and second image, based on the colors assigned to each channel. For every chromosome, the first and second dots indicate the color of that chromosome pair in the first and second image, respectively. This chart can be used to visually "color-karyotype" all chromosomes.Because BIO was detected with AMCA and was visualized in the first image, this coloring chart was named 3+2, meaning that chromosomes were labeled with 3 colors in the first image and two colors in the second. Image pairs i, j, and k, l depict the first and second images of two CCK analyses of normal metaphases using the 2+3 algorithm #4 in table 2. Images i, j were captured using a cooled CCD camera whereas images k, l were captured with an RGB digital photographic camera (Olympus DP-10). In i and k FITC is pseudocolored green, Texas-Red red and AMCA (BIO) blue. In j and l, FITC (DNP) was pseudocolored green and Texas-Red (DIG) red, and thus chromosomes were only green, red or yellow (green + red). The faint blue color of some chromosomes comes from DAPI counterstaining. When the green, red and blue channels were combined, the DAPI channel was shown only at a reduced lightness, in order to avoid interference with the red and green hybridization colors.
Signal strength and discrimination
The use of only three fluorescent dyes, showing little or no interference with one another, increases signal discrimination. Use of labeled antibodies, carrying 2-4 dye molecules/protein molecule, yields much stronger signals compared to labeled nucleotides only. Both factors may make CCK a more robust procedure for detecting small translocated chromosome fragments, when compared with M-FISH or SKY.
Each antibody detection step requires 5 minutes incubation followed by 10 minutes of washing. After first image capturing, removal of antifade/mounting media requires 10-15 minutes washing. As metaphase coordinates are already known, "second image" capturing is rapid, at a rate of about 15 metaphases every 30-45 minutes.
System requirements and image processing
Image capturing can be performed with either digital photographic or video/CCD cameras, using a fluorescence microscope equipped with three or four filters. Image processing (correction of image shift between the first and second images; background subtraction; pseudocoloring; karyotyping) can be performed with but does not require commercial FISH or M-FISH packages. The grayscale images composing the first and the second CCK images can be processed using generic image-processing software (Adobe Photoshop). Although Photoshop does not include a karyotyping feature, combinatorial labeling with three dyes yields only seven colors, very easy to discriminate by the human eye. Thus visual chromosome identification becomes a simple task, using color charts (Fig. 3 and 4). The rapidly expanding field of digital photographic cameras, makes it possible to replace "classic" film-cameras with digital cameras on virtually any microscope. Images captured in the red, green and blue channels are stored on a diskette or memory chip and subsequently transferred to any computer platform for further processing (Fig. 4k and l).