1 FISH Techniques, FISH Probes and Their Applications in Medicine and Biology - An Overview
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ratio that identifies the probe (Nederlof et al. 1992). For example five different ratios ofred and green fluorescence attached to five different probes
allow the discrimination of five different targets with only two fluorochromes. By the recently developed COBRA (Combined Binary Ratio labeling) technique the two methods can be combined and then up to 100
label types can be obtained (Tanke et al. 1999).
The developments of new FISH-techniques have been paralleled by
developments in the microscope systems used. While the basic microscopic features have been rather constant, we have witnessed an impressing development in fIlter technology, where it is now possible to order very
precise custom-designed fIlters. This has, in connection with the development of many new fluorescent dyes, made it possible to perform extensive
multicolor analyses. New fluorescent dyes and fIlters have extended the
potential of FISH by simultaneously detecting multiple genomic targets
in different colors. Thus, the availability of an increasing number of spectrally distinct fluorochromes can be utilized for the direct or indirect labeling of nucleic acid probes, and then more targets can be visualized in
parallel by simply mixing differently labeled probes for a given target.
Fluorescent signals can be viewed both directly in the fluorescence microscope or by using a CCD-camera (charged coupled devices) coupled to
the microscope and connected to a computer. Images from each filter set
are then captured followed by image analysis in specifically designed software. If the signal to noise ratio is too low, either because of high background or low intensity of signals, the image analysis software can improve the ratio. The images which are in the gray scale can then be overlaid
and given artificial colors for presentation. Confocal microscopy utilizes a
laser for excitation and photomultipliers for detection of emitted light.
Specialized software control the laser, photomultipliers and have similar
possibilities for image analysis. One important advantage of using confocal microscopy is that different confocal planes can be captured and processed to generate a three-dimensional image. Such an approach allows
for an improved analysis of nuclei or tissues which often contain overlapping signals. However, these analyses are impractical in routine diagnostics at the present technological stage.
24-color karyotyping by spectral analysis (SKY) and multifluor FISH (mFISH)
One of the most recent refinements in FISH-based multicolor techniques
for screening whole genomes for both structural and numerical chromosomal aberrations are the following two technically related innovations:
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ratio that identifies the probe (Nederlof et al. 1992). For example five different ratios ofred and green fluorescence attached to five different probes
allow the discrimination of five different targets with only two fluorochromes. By the recently developed COBRA (Combined Binary Ratio labeling) technique the two methods can be combined and then up to 100
label types can be obtained (Tanke et al. 1999).
The developments of new FISH-techniques have been paralleled by
developments in the microscope systems used. While the basic microscopic features have been rather constant, we have witnessed an impressing development in fIlter technology, where it is now possible to order very
precise custom-designed fIlters. This has, in connection with the development of many new fluorescent dyes, made it possible to perform extensive
multicolor analyses. New fluorescent dyes and fIlters have extended the
potential of FISH by simultaneously detecting multiple genomic targets
in different colors. Thus, the availability of an increasing number of spectrally distinct fluorochromes can be utilized for the direct or indirect labeling of nucleic acid probes, and then more targets can be visualized in
parallel by simply mixing differently labeled probes for a given target.
Fluorescent signals can be viewed both directly in the fluorescence microscope or by using a CCD-camera (charged coupled devices) coupled to
the microscope and connected to a computer. Images from each filter set
are then captured followed by image analysis in specifically designed software. If the signal to noise ratio is too low, either because of high background or low intensity of signals, the image analysis software can improve the ratio. The images which are in the gray scale can then be overlaid
and given artificial colors for presentation. Confocal microscopy utilizes a
laser for excitation and photomultipliers for detection of emitted light.
Specialized software control the laser, photomultipliers and have similar
possibilities for image analysis. One important advantage of using confocal microscopy is that different confocal planes can be captured and processed to generate a three-dimensional image. Such an approach allows
for an improved analysis of nuclei or tissues which often contain overlapping signals. However, these analyses are impractical in routine diagnostics at the present technological stage.
24-color karyotyping by spectral analysis (SKY) and multifluor FISH (mFISH)
One of the most recent refinements in FISH-based multicolor techniques
for screening whole genomes for both structural and numerical chromosomal aberrations are the following two technically related innovations: