20
EIGIL KJELDSEN and STEEN K0LVRAA
SKY (Schrock et al. 1996) and mFISH (Speicher et al. 1996). Both methods
rely on metaphases and allow simultaneous identification of different
chromosomes, with each chromosome displayed in a characteristic color.
In both methods all 24 differently labeled human chromosomes are hybridized in a single hybridization experiment. With five fluorescent colors
at hand, enough combinations of these are available to give 24 artificial
colors in the final presentation. The methods of detecting these combinations are, however, different in the two systems.
mFISH is a fIlter-based approach and employs the sequential image
acquisition using five different fluorochrome-specific optical fIlters,
each ofwhich are narrow band pass fIlters that allow maximum distinction
of the fluorescent dyes. Special image analysis software calculates a chromosome segmentation mask based on the DAPI image, followed by measuring the intensities in these predefined areas for all subsequently acquired images. Before these calculations, the software performs an image
shift correction. Based on the labeling schemes the program decides
whether a single pixel contains signals from one or more fluorochromes
and this information forms the basis for chromosome identification and
subsequent color assignment.
SKY is based on spectral imaging which is a combination of spectroscopy, CCD imaging and fluorescence microscopy. An interferometer
coupled to a CCD camera generates a fluorochrome specific optical
path difference that, in turn can be Fourier transformed to provide spectral information.
Consequently, the fluorescence emission spectrum can be recovered
simultaneously at all image points with one exposure. Specialized software
then classifies the images by identifying pixels with identical spectra and
then assigning these pixels the same classification color, which in turn is
the basis for the actual karyotyping.
The probe sets for SKY and mFISH are commercially available at a relatively high cost but recently Roberts et al. (1999) have presented new probe
construction protocols and strategies for multi-color karyotyping.
Bar-coding of human chromosomes
By using subregional DNA probes in different colors, a pattern that has
been termed "chromosome bar code" can be produced. This enables chromosome identification by producing a limited number of bars on each
chromosome (Lengauer et al. 1992), providing a potential to screen for
intra-chromosomal rearrangements (e.g. deletions and inversions).
EIGIL KJELDSEN and STEEN K0LVRAA
SKY (Schrock et al. 1996) and mFISH (Speicher et al. 1996). Both methods
rely on metaphases and allow simultaneous identification of different
chromosomes, with each chromosome displayed in a characteristic color.
In both methods all 24 differently labeled human chromosomes are hybridized in a single hybridization experiment. With five fluorescent colors
at hand, enough combinations of these are available to give 24 artificial
colors in the final presentation. The methods of detecting these combinations are, however, different in the two systems.
mFISH is a fIlter-based approach and employs the sequential image
acquisition using five different fluorochrome-specific optical fIlters,
each ofwhich are narrow band pass fIlters that allow maximum distinction
of the fluorescent dyes. Special image analysis software calculates a chromosome segmentation mask based on the DAPI image, followed by measuring the intensities in these predefined areas for all subsequently acquired images. Before these calculations, the software performs an image
shift correction. Based on the labeling schemes the program decides
whether a single pixel contains signals from one or more fluorochromes
and this information forms the basis for chromosome identification and
subsequent color assignment.
SKY is based on spectral imaging which is a combination of spectroscopy, CCD imaging and fluorescence microscopy. An interferometer
coupled to a CCD camera generates a fluorochrome specific optical
path difference that, in turn can be Fourier transformed to provide spectral information.
Consequently, the fluorescence emission spectrum can be recovered
simultaneously at all image points with one exposure. Specialized software
then classifies the images by identifying pixels with identical spectra and
then assigning these pixels the same classification color, which in turn is
the basis for the actual karyotyping.
The probe sets for SKY and mFISH are commercially available at a relatively high cost but recently Roberts et al. (1999) have presented new probe
construction protocols and strategies for multi-color karyotyping.
Bar-coding of human chromosomes
By using subregional DNA probes in different colors, a pattern that has
been termed "chromosome bar code" can be produced. This enables chromosome identification by producing a limited number of bars on each
chromosome (Lengauer et al. 1992), providing a potential to screen for
intra-chromosomal rearrangements (e.g. deletions and inversions).