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Automated Signal Extraction
Automated Signal Extraction
Introduction
This topic will show you how to set-up a measurement program using the Image
Analysis Wizard. After this the program will be used to count the number of
fluorescence spots in a multichannel image. In this example we are using a
multichannel image with 2 channels (1st channel blue, DAPI / 2nd channel green,
GFP) of fluorescence-stained cell nuclei. First we detect the blue-stained cell nuclei
in the first channel and then the green stained signals in the second channel. Then
we measure the number of green fluorescence signals per nucleus.
3 Click on the New entry and enter a name for your measurement program.
The Image Analysis Wizard is opened. In the left-hand area you will see 7 steps
which will guide you through the wizard.
Step 1: Classes
Procedure 1 Click on Classes1 in the list and enter Nuclei in the Name input field.
2 Select a blue color from the dropdown list in the Color section.
3 Click on the Class1 entry in the list and enter Individual Nucleus in the Name
input field.
2 Click on Classes3 in the list and enter Signals in the Name input field.
4 Click on the Class3 entry in the list and enter Individual Signal in the Name
input field.
You have now setup a class pair (child class) for the signals inside the Individual
Nucleus class (parent class).
2 Click on Next.
2 In the Smooth section select Gauss from the dropdown list and set the
parameter Sigma to 1.5.
The detected nuclei are overlaid in blue. The threshold values are displayed in
the Threshold section in the Lower / Upper input fields.
4 Click on the areas of the blue cell nuclei that have not yet been detected until
these have been completely overlaid.
5 Select the Watersheds entry from the dropdown list in the Separate section
and set the number to 17.
Clear separation lines are now visible between the cell nuclei.
7 In the Smoothing section select Gauss from the dropdown list and set the
parameter Sigma to 1.5.
The detected signals are overlaid in green. The threshold values are displayed in
the Threshold section in the Lower / Upper input fields.
9 Click on the areas of the green signals that have not yet been detected until
these have been completely overlaid.
12 In the Separate section select the Watersheds entry from the dropdown list
and set the number to 17.
13 Click on Next.
Step 4: Condition
2 Click on Next.
2 Click on Next.
The features are displayed in the Selected Features list on the left.
4 Remove superfluous features from the list. Select the feature and click on the
Delete button .
The features are displayed in the Selected Features list on the left.
9 Remove superfluous features (e.g. Area, Perimeter) from the list. Select the
feature and click on the Delete button .
The features are displayed in the Selected Features list on the left.
14 Remove superfluous features from the list. Select the feature and click on the
Delete button .
18 Remove superfluous features (e.g. Area, Perimeter) from the list. Select the
feature and click on the Delete button .
20 Click on Next.
Step 7: Measurement
The number of measured nuclei is displayed in the data table to the right of the
image.
The object ID of the measured nuclei is displayed in the data list to the right of
the image.
The ID of the parents (corresponds to the ID of the nucleus) and the number of
measured signals are displayed in the data table to the right of the image.
¢ You have loaded the measurement program that you have generated.
The Analysis View now also appears in the Center Screen Area.
In the Analysis View you will see your image with the measured cell nuclei
overlaid in blue and the signals overlaid in green. Right of this, the data list
containing the number of signals per nucleus.
Only the number of signals of measured nuclei is displayed. Nuclei touching the
frame are not taken into account.
Procedure 1 Click on the Create Measurement Data Table button on the Analysis tab.
The two data lists are now separate documents and have been removed from
the image.
2 Save each of the data lists via the File menu | Save As. Allocate a name and
select .csv as the file type.
The measurement data tables are saved in CSV format and can therefore be
opened directly in Excel.
3 Click on the image and save it via the File menu | Save As. Allocate a name
and select .czi as the file type.
The image is saved with the measurement results. If you open the image, the
measurement results can be viewed in the Analysis View.