Table 3 Parameters used for automated scanning

Capturing

Color Channels

Max. Integration Time

For capturing images with comparable signal intensities, automatic integration time adjustment was used to reach a certain saturation level in the images while the maximum integration time was limited to 0.5 s (green) and 0.33 s (red) for keeping the background level at low intensities for empty image fields (e.g. not showing signals).

Saturation Area

N Focus Planes

Due to the fact that nuclei are not perfectly flattend on the glass slide by preparation but show Z dimensions within a certain range, the fluorescently labeled chromosomes may be randomly localized in the nucleus also in Z direction.

Distance

CCD Gain

A CCD camera gain factor was specified to reduce the integration times needed and thereby increase the scanning speed. With the value specified the electronic noise in the captured images was still negligible.

Use CS Mask during Capt

This parameter was activated to use the counterstain mask for integration time adjustment. As bright artifacts within the image field would usually interfere with the automatic integration time adjustment, using the counterstain mask enabled correct adjustment for image fields where such artifacts were only present outside the nuclei.

Image Processing

An image processing operation was applied to the signal channels to reduce the noise level without significantly reducing the sharpness of the image by vertical median filtering. This filtering was used to remove small "hot spots" of one pixel size in the images which appear in CCD camera images after long integrations or due to camera pixel defects.

Cell Selection

Obj. Threshold

An object threshold of 23% in the counterstain channel was used to segment the cell nuclei. The value is a percentage based on the total contrast range of the captured image.

Min. Nucleus Area

The minimum/maximum area in μ m² for a single cell nucleus to be accepted for analysis was used e.g. to exclude (larger) cell clusters.

Max. Nucleus Area

Max. Rel. Conc. Depth

This criterion has been used to discriminate single cells (showing a convex contour with only small concave areas) from cell clusters (which usually have large concavities). The limit is specified relative to the nucleus diameter.

Max. Aspect Ratio

This criterion has been used to discriminate the nuclei of interest from more elongated objects. It specifies the maximum ratio of the nucleus diameters along the long and the short principal axis.

Cell Processing

CS/R/G

Additional image processing was applied to reduce background/exclude image content outside nucleus contour.

Extend CS Mask

To correctly identify signals on the nucleus edge the counterstain mask has been extended by 0.5 μ m.

Features/Spot Counting

Max. Spot Rel. Area

To differentiate true FISH spots from variations in the fluorescence background, an upper limit for the relative area of a spot, compared to the whole nucleus (in units of 1/1000) was defined, This was mainly of interest for the green channel (Y chromosome).

R (X)

SpotCounts (5,27)

The number of red FISH spots was determined. Spots were accepted (counted) if they had a minimum distance of 0.5 μ m and a minimum intensity of 27% compared to the brightest spot in the same cell.

Reject if > 2

Cells with more than 2 red spots were automatically rejected.

G (Y)

SpotCounts (14,78)

The number of green FISH spots was determined. Spots were accepted (counted) if they had a minimum distance of 1.4 μ m and a minimum intensity of 78% compared to the brightest spot in the same cell.

Reject if > 2

Cells with more than 2 green spots are automatically rejected.

Reject if No Spots

Cells not showing any X signals are automatically rejected.