12 Chromosome Analysis by Fluorescence in situ Hybridization
183
propriate fllter sets. For certain experiments, the use of filter sets selective
for a particular fluorochrome is preferred. Other applications require the
simultaneous visualization of different fluorochromes which can be
achieved by the use of multi-band pass filters. These may be advantageous
for the evaluation of multi-color experiments. They also provide a solution
to the so-called registration problem: when several images of one object are
taken (by using single bandpass filters) and overlayed to obtain a combined
image, more or less pronounced shifts are detected, which are based on
small differences in the optical pathway due to variations in filter and mirror positions. Advanced applications of FISH rely on the quantitative analysis of fluorescence intensities: multicolor hybridization by ratio labelling
(see above) and, in particular, the powerful approach of comparative genomic hybridization ( CGH; Kallioniemi et al. 1992 ). For the description of the
CGH approach, we refer the interested reader to the Iiterature (Kallioniemi
et al. 1992; Du Manoir et al. 1993; Joos et al. 1993; Forozan et al. 1997). For
such applications optical detection systems are needed which record signal
intensities in a linear mode over a wide range of intensities. Cooled CCD
(charge-coupled device, (Hiraoka et al. 1987) cameras provide powerful
tools for this application due to their wide dynamic range. They are currently the most sensitive systems and, thus, are also very useful for the anaIysis of weak signals, e.g. obtained using very small probes (smaller than 2Skb). Although confocallaser scanning microscopy is also capable of sensitive digital imaging, this procedure is primarily designed for three-dimensional analyses of fluorescently labeled specimens. Moreover, the use of
fluorochromes is limited to those which are excited by the Iasers available
in the instrument. In addition, confocallaser microscopes are much more
expensive than the most sensitive camera systems. Therefore, for two-dimensional analyses, cooled CCD cameras are preferred. When purchasing
equipment for digital imaging microscopy, attention has to be paid to both
the memory and storage capacity of the hardware as well as to the software
that is available for special applications.
References
Anastasi J, Yardiman JW, Rudinsky R, Patel M, Nachman J, Rubin CM, LeBeau MM
(1991) Direct correlation of cytogenetic findings with cell morphology using in
situ hybridization: an analysis of suspicious cells in hone marrow specimens of
two patients completing therapy for acute lymphoblastic leukemia. Blood 77:
2456-2462
Bentz M, Döhner H, Cabot G, Lichter P (1994) Fluorescence in situ hybridization in leukemias: The FISH are spawning! Leukemia 8: 1447-1452
183
propriate fllter sets. For certain experiments, the use of filter sets selective
for a particular fluorochrome is preferred. Other applications require the
simultaneous visualization of different fluorochromes which can be
achieved by the use of multi-band pass filters. These may be advantageous
for the evaluation of multi-color experiments. They also provide a solution
to the so-called registration problem: when several images of one object are
taken (by using single bandpass filters) and overlayed to obtain a combined
image, more or less pronounced shifts are detected, which are based on
small differences in the optical pathway due to variations in filter and mirror positions. Advanced applications of FISH rely on the quantitative analysis of fluorescence intensities: multicolor hybridization by ratio labelling
(see above) and, in particular, the powerful approach of comparative genomic hybridization ( CGH; Kallioniemi et al. 1992 ). For the description of the
CGH approach, we refer the interested reader to the Iiterature (Kallioniemi
et al. 1992; Du Manoir et al. 1993; Joos et al. 1993; Forozan et al. 1997). For
such applications optical detection systems are needed which record signal
intensities in a linear mode over a wide range of intensities. Cooled CCD
(charge-coupled device, (Hiraoka et al. 1987) cameras provide powerful
tools for this application due to their wide dynamic range. They are currently the most sensitive systems and, thus, are also very useful for the anaIysis of weak signals, e.g. obtained using very small probes (smaller than 2Skb). Although confocallaser scanning microscopy is also capable of sensitive digital imaging, this procedure is primarily designed for three-dimensional analyses of fluorescently labeled specimens. Moreover, the use of
fluorochromes is limited to those which are excited by the Iasers available
in the instrument. In addition, confocallaser microscopes are much more
expensive than the most sensitive camera systems. Therefore, for two-dimensional analyses, cooled CCD cameras are preferred. When purchasing
equipment for digital imaging microscopy, attention has to be paid to both
the memory and storage capacity of the hardware as well as to the software
that is available for special applications.
References
Anastasi J, Yardiman JW, Rudinsky R, Patel M, Nachman J, Rubin CM, LeBeau MM
(1991) Direct correlation of cytogenetic findings with cell morphology using in
situ hybridization: an analysis of suspicious cells in hone marrow specimens of
two patients completing therapy for acute lymphoblastic leukemia. Blood 77:
2456-2462
Bentz M, Döhner H, Cabot G, Lichter P (1994) Fluorescence in situ hybridization in leukemias: The FISH are spawning! Leukemia 8: 1447-1452