29 Spectral Imaging Analysis of Metaphase and Interphase Cells
379
Procedure
Building the combinatorial table file is the first step of the spectral karyotyping. Acquire the fluorescencence spectra of pure dyes using the SKY1 filter set and build the combinatorial table file (CTB) (also refered to as
'reference spectral library') to be used in all subsequent measurements.
1. For spectral karyotyping on metaphase spreads, five pure fluorochromes (Spectrum Green, Spectrum Orange, Texas Red, Cy5,
Cy5.5) have been acquired by the SD200 SpectraCube spectral imaging
system and the CTB files for human and mouse karyotyping have been
set up in the software.
2. For interphase cell analysis, acquire the spectrum of pure fluorochromes (Spectrum Green, FITC, Spectrum Orange, Cy3, Cy5, and
Cy5.5).
3. The CTB file for interphase cell analysis is built according to the fluorochrome-labeling scheme shown in Table 1.
4. A pseudo-color for each target in the CTB table is assigned to all points
in the image.
Select a target of interest, acquire the image through the SKY-1 filter
(saved as a raw file) and DAPI filter (saved as a tif file).
1. In the Sky View software, open the spectrum image file, dapi file, and
CTB file.
2. The image acquired through the SKY-1 filter is displayed by assigning
red, green or blue colors (RGB pseudo-color image) to three ranges of
interest in the spectrum (usually 450-550, 550-650, 650-850 nm).
3. In the RGB pseudo-color image, delineate the signal domain by drawing contour lines around each signal (interphase cells analysis) or each
chromosome (metaphase cells analysis).
4. Based on the measurement of the spectrum for each signal or metaphase, a spectral classification algorithm is applied that allows the assignment of a classification pseudo-color to all points in the image that
had the same spectrum. This algorithm forms the basis for chromosome identification by spectral karyotyping.
Build combinatorial
table (aB) file
Acquiring the RGB
color and DAPI
images
Data analysis
379
Procedure
Building the combinatorial table file is the first step of the spectral karyotyping. Acquire the fluorescencence spectra of pure dyes using the SKY1 filter set and build the combinatorial table file (CTB) (also refered to as
'reference spectral library') to be used in all subsequent measurements.
1. For spectral karyotyping on metaphase spreads, five pure fluorochromes (Spectrum Green, Spectrum Orange, Texas Red, Cy5,
Cy5.5) have been acquired by the SD200 SpectraCube spectral imaging
system and the CTB files for human and mouse karyotyping have been
set up in the software.
2. For interphase cell analysis, acquire the spectrum of pure fluorochromes (Spectrum Green, FITC, Spectrum Orange, Cy3, Cy5, and
Cy5.5).
3. The CTB file for interphase cell analysis is built according to the fluorochrome-labeling scheme shown in Table 1.
4. A pseudo-color for each target in the CTB table is assigned to all points
in the image.
Select a target of interest, acquire the image through the SKY-1 filter
(saved as a raw file) and DAPI filter (saved as a tif file).
1. In the Sky View software, open the spectrum image file, dapi file, and
CTB file.
2. The image acquired through the SKY-1 filter is displayed by assigning
red, green or blue colors (RGB pseudo-color image) to three ranges of
interest in the spectrum (usually 450-550, 550-650, 650-850 nm).
3. In the RGB pseudo-color image, delineate the signal domain by drawing contour lines around each signal (interphase cells analysis) or each
chromosome (metaphase cells analysis).
4. Based on the measurement of the spectrum for each signal or metaphase, a spectral classification algorithm is applied that allows the assignment of a classification pseudo-color to all points in the image that
had the same spectrum. This algorithm forms the basis for chromosome identification by spectral karyotyping.
Build combinatorial
table (aB) file
Acquiring the RGB
color and DAPI
images
Data analysis