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Chapter 5: Configuring Plate Acquisition Options
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Calculating the Intensity Above Local Background Gray Levels
The Sites to Visit tab Adaptive Acquisition option is a computational algorithm that analyzes
the number of cells on the fly during sample acquisition. This increases the chances of
collecting valid data in every well. The number of sites the software acquires per well is based
on the number of cells per well.
To calculate the intensity above local background gray levels value:
1. If the Region Tools toolbar does not display, do one of the following:
From the simplified menu, click Measure > Regions > Regions Tools.
From the standard menu, click Regions > Regions Tools.
Figure 5-12: Region Tools Toolbar
2. Click .
3. Open the source image.
4. Move the cursor over the image and watch the status bar at the bottom of the software
window to note the X, Y coordinates and the gray level value of the pixel that is under the
cursor. The X and Y coordinates are enclosed in parentheses and the gray level value
displays to the right. For example, (48, 158) -> 18000 indicates that 18000 is the average
gray level value of the pixel.
5. Locate one of the dimmest objects (for example, a nucleus, if applicable) that you want the
software to detect.
6. Position the cursor on the dimmest part of the object and make note of the gray level
value that displays in the status bar.
Figure 5-13: Status Bar Gray Level Indicators
7. Position the cursor just to the outside of the object and make note of the gray level value
that displays in the status bar. For example, (48, 162) -> 2000 indicates that 2000 is the
average gray level value of the background just outside the object.
Tip: Draw a line region across a cell and its local background, and then use the
Linescan tool to see more exact intensity values. To access the Linescan tool, click
Measure > Intensities > Linescan.
8. Subtract the gray level value of the background from the gray level value of the object
and enter one quarter to one half of this value in the Intensity Above Local Background
field.
9. To determine if the acquisition settings are appropriate to differentiate nuclei, do the
following:
a. Select a well and site to review your settings.
b. Click Focus to bring the sample into focus.
Note: Focus does a large-range autofocus on the selected well and site. To
ensure that your cell count settings are accurate, bring the sample into focus
before you click Test Segmentation.
c. Click Test Segmentation to snap an image and count the nuclei. Both an acquisition
image and the total number of cells that were counted in the image display.
10. Repeat the previous steps until your test results are satisfactory.