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Measuring Mean Fluorescence Intensity on a Ring around the Primary Object

The following example shows how to use the Zone of Influence (ZOI) method to measure intensities within a ring that is associated to the main object, e.g. the cell nucleus. An application example are transport assays where the intensities of a certain fluorescent marker in the cytoplasm are compared to the intensities within the nucleus.

In this example we use a multichannel image of fluorescence-stained cells. The cell nuclei are stained with AF568 and the mitochondria are stained with AF488. First, we detect the nuclei in the AF568-channel as primary object. A zone of influence is generated around each detected primary object. In this area, we can define a ring and specify its thickness and distance from the main object. You can use this ring to measure intensities or to detect further sub-objects on it. For more information, see Counting the Number of Objects in a Ring around the Nucleus.

Step 3: Automatic Segmentation

  1. Select Primary Object and set up suitable parameters to detect the objects, i.e. threshold, area, separation.
  2. As soon as objects are detected, the ZOI and Ring are automatically created around each primary object with the preset parameters.
  3. To modify Ring Distance and Width, select the Ring Element class.
  4. Now, you can define the location and dimension of the ring flexibly. You can set it at the edge of the main object or inside the main object. You can also define an arbitrary distance.
  5. Define the following parameters:
  6. Ring Distance: Distance from surface of the primary object. Negative values means that the ring starts at the defined distance within the primary object. Ring Width: Defines the width of the ring.
  7. The ZOI is automatically adapted to exceed the class with the larger diameter, i.e. either ring or primary object, by at least 3 pixels (default setting).
Ring distance: 0 pixel; Ring width: 10 pixel
Ring distance: 0 pixel; Ring width: 10 pixel
Ring distance: 5 pixel; Ring width: 10 pixel
Ring distance: 5 pixel; Ring width: 10 pixel
Ring distance: -5 pixel; Ring width: 5 pixel
Ring distance: -5 pixel; Ring width: 5 pixel

Modifying the ZOI Width

  1. Select ZOI-class, and with the ZOI Width slider, set the distance. You can set the distance between the outer border of the ZOI and the outer border of either ring or primary object, respectively. The ZOI Width is at least 3 pixels larger than either the ring or the primary object, whichever is larger. The ZOI area incorporates also the area of the ring and the primary object, and thus can serve for example for measuring features over the complete cell.
ZOI Width set to 30 pixel
ZOI Width set to 30 pixel

Step 6: Result Preview

In this step you see a preliminary result of the image analysis.

  1. Click on the different objects in the Analysis tab to get the preliminary measurement result for all objects.
  2. Click Finish, to save the analysis settings and close the wizard.
  3. The wizard closes. The analysis settings are saved.

Executing the Analysis Setting

You have the following options to run a predefined image analysis setting on your data set:

  • Start Interactive Analysis: Analyze interactively with all steps that have been selected with the checkbox Interactive during setup of the image analysis.
  • Start Analysis: Runs the image analysis setting without dialog.

When the analysis is finished, the main view switches to the Analysis tab and displays the segmented image along with the results of the analysis.

Select the different objects to display the corresponding measurement tables. The data in the tables and the regions in the image are interlinked. A click on the object in the image highlights the corresponding line in the data table and vice versa.

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