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HORST ZITZELSBERGER, BENJAMIN O'BRIEN, and HEINZ-ULRICH WEIER
Image acquisition
Images of the in situ hybridization results are recorded, for example on the
Quantitative Image Microscope System (QUIPS) using the Scilimage software (Weier et aI, 1995b) or on the MetaSystems image analysis system
(see Chapter 2, Microscopy and Imaging Systems by Andreas Plesch). The
image analysis systems are attached to a fluorescence microscope e.g., a
Zeiss Axioplan 2 fluorescence microscope equipped with single-band excitation filters for DAPI, FITC, TRITC, CyS, a motorized filter changer and
oil immersion objectives (40 x, 63 x). For visual evaluation, triple and
quadruple-color filter sets are recommended.
Results
RxFISH
Figure 1A shows a normal Rx-FISH pattern on human metaphase chromosomes. After in vitro irradiation of peripheral lymphocytes with 3 Gy Xrays and 1.2 Gy fission neutrons, para- and pericentric inversions became
apparent due to atypical banding patterns (Fig. 1B, 1C). Breakpoints were
determined by use of DAPI-banded images. In this way, inversions could
be reliably quantified either for the whole chromosome complement or
selected chromosomes. For an estimation of the LET dependent ratio between inversion and symmetrical translocations, the latter must be scored
either in a separate three-color FISH painting experiment or by spectral
karyotyping for the whole chromosome complement.
CR
On a normal metaphase chromosome 10, our scheme begins (from pter to
qter) with a probe visualized in red at the distal short arm of chromosome
10, followed by a probe detected in infrared, next a probe seen in green,
and then one in yellow. The next probe, detected in red, started a new cycle
of colors (Fig. 2) creating a pattern of a chromosome-specific
probes prepared from PI clones (Table I), which provided further clues
for the identification of marker chromosomes. A cytogenetic alteration is
suspected, if the normal color sequence and signal pattern is altered. As an
example, we applied a CR 10 set to metaphase spreads of the cell line TPC-
HORST ZITZELSBERGER, BENJAMIN O'BRIEN, and HEINZ-ULRICH WEIER
Image acquisition
Images of the in situ hybridization results are recorded, for example on the
Quantitative Image Microscope System (QUIPS) using the Scilimage software (Weier et aI, 1995b) or on the MetaSystems image analysis system
(see Chapter 2, Microscopy and Imaging Systems by Andreas Plesch). The
image analysis systems are attached to a fluorescence microscope e.g., a
Zeiss Axioplan 2 fluorescence microscope equipped with single-band excitation filters for DAPI, FITC, TRITC, CyS, a motorized filter changer and
oil immersion objectives (40 x, 63 x). For visual evaluation, triple and
quadruple-color filter sets are recommended.
Results
RxFISH
Figure 1A shows a normal Rx-FISH pattern on human metaphase chromosomes. After in vitro irradiation of peripheral lymphocytes with 3 Gy Xrays and 1.2 Gy fission neutrons, para- and pericentric inversions became
apparent due to atypical banding patterns (Fig. 1B, 1C). Breakpoints were
determined by use of DAPI-banded images. In this way, inversions could
be reliably quantified either for the whole chromosome complement or
selected chromosomes. For an estimation of the LET dependent ratio between inversion and symmetrical translocations, the latter must be scored
either in a separate three-color FISH painting experiment or by spectral
karyotyping for the whole chromosome complement.
CR
On a normal metaphase chromosome 10, our scheme begins (from pter to
qter) with a probe visualized in red at the distal short arm of chromosome
10, followed by a probe detected in infrared, next a probe seen in green,
and then one in yellow. The next probe, detected in red, started a new cycle
of colors (Fig. 2) creating a pattern of a chromosome-specific
probes prepared from PI clones (Table I), which provided further clues
for the identification of marker chromosomes. A cytogenetic alteration is
suspected, if the normal color sequence and signal pattern is altered. As an
example, we applied a CR 10 set to metaphase spreads of the cell line TPC-