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BURT G. FEUERSTEIN et al.
erochromatic regions of chromosomes 1,9, and 16. (2) The ratio of the
average foreground signal (within the segmentation mask) to the average
background signal (outside of the mask) should be >1.5 for each fluorochrome. (3) The coefficient of variation for foreground intensity of the
reference DNA image should be <20%. (4) Granular hybridizations
should be avoided. Granularity produces rapid changes in intensity in
foreground signal. (5) Only well-separated and straight chromosomes
should be selected for analysis.
Data analysis The software must perform a number of functions that analyze the data
collected by the CCD camera (14). Segmentation separates the image into
regions that define the chromosomes as foreground. This is often done by
thresholding the reference and DAPI images. Various routines are used to
remove nuclei, separate chromosomes and refine chromosome edges.
Normalization equalizes the median intensities of the test and the reference fluorescence intensities by setting each equal to one. This is done by
subtracting an estimate ofthe background around each chromosome from
each pixel in the chromosome to determine the corrected foreground intensities. The median intensity for the metaphase is determined from the
corrected foreground intensities, and the inverse of that value is used to
normalize the fluorescence intensity for each pixel in the metaphase foreground. Since the test and reference fluorescence intensities are each normalized to one, CGH can only detect relative values for alterations in copy
number. Figure 2 shows the normalized image for microdissected and
DOP-PCR amplified material from a grade II astrocytoma with -lp and
-19q. Notice absence of red or green intensity at centromeres and the relatively high red intensity at Ip and 19q (arrows). Ratio profile. The normalized fluorescence intensities are integrated along slices orthogonal to
the axis of each chromosome. Dividing the integrated test and reference
values, point by point along each chromosome, defines the ratio profile.
Average ratio profiles. The lengths of profiles for each chromosome are
normalized to a standard length (15). Then the average fluorescence ratio
and its variation among the individual ratio profiles are determined and
displayed at each point along the chromosome. Figure 3 is the average
ratio profile derived in part from the data shown in Fig. 2. There is
loss on 1p and 19q. Note the relatively tight standard deviations.
BURT G. FEUERSTEIN et al.
erochromatic regions of chromosomes 1,9, and 16. (2) The ratio of the
average foreground signal (within the segmentation mask) to the average
background signal (outside of the mask) should be >1.5 for each fluorochrome. (3) The coefficient of variation for foreground intensity of the
reference DNA image should be <20%. (4) Granular hybridizations
should be avoided. Granularity produces rapid changes in intensity in
foreground signal. (5) Only well-separated and straight chromosomes
should be selected for analysis.
Data analysis The software must perform a number of functions that analyze the data
collected by the CCD camera (14). Segmentation separates the image into
regions that define the chromosomes as foreground. This is often done by
thresholding the reference and DAPI images. Various routines are used to
remove nuclei, separate chromosomes and refine chromosome edges.
Normalization equalizes the median intensities of the test and the reference fluorescence intensities by setting each equal to one. This is done by
subtracting an estimate ofthe background around each chromosome from
each pixel in the chromosome to determine the corrected foreground intensities. The median intensity for the metaphase is determined from the
corrected foreground intensities, and the inverse of that value is used to
normalize the fluorescence intensity for each pixel in the metaphase foreground. Since the test and reference fluorescence intensities are each normalized to one, CGH can only detect relative values for alterations in copy
number. Figure 2 shows the normalized image for microdissected and
DOP-PCR amplified material from a grade II astrocytoma with -lp and
-19q. Notice absence of red or green intensity at centromeres and the relatively high red intensity at Ip and 19q (arrows). Ratio profile. The normalized fluorescence intensities are integrated along slices orthogonal to
the axis of each chromosome. Dividing the integrated test and reference
values, point by point along each chromosome, defines the ratio profile.
Average ratio profiles. The lengths of profiles for each chromosome are
normalized to a standard length (15). Then the average fluorescence ratio
and its variation among the individual ratio profiles are determined and
displayed at each point along the chromosome. Figure 3 is the average
ratio profile derived in part from the data shown in Fig. 2. There is
loss on 1p and 19q. Note the relatively tight standard deviations.