Concentric Layers Protocol

Introduction

The Concentric Layers protocol is designed to measure the layer thickness of concentric layers around a central core/area.

Concentric Layers Protocol Information:

• Inputs: One image containing the sample of interest. 
• Time: End Point 
• Data Output: 
  • Layer Measurements: Thickness (Mean), Thickness (Standard Deviation), Thickness (Max) and Thickness (Min).
  • Optional snapshot image of the analyzed location(s).

Concentric Layer Protocol Setup: Step 1

For additional information on setting up protocols, please refer to the Protocols Help section: Protocol Setup.

1. Load your image dataset. An example of a dataset suitable for concentric layers analysis can be found in the File/Open/Demo Images in the Protocol Materials/Concentric Layers folder. The following instructions are based on using the demonstration image.

2. Select the Count/Size tab.
3. Press the Protocols button to display the protocol browser.
4. Load the default Concentric Layers protocol located in the Materials collection
5. The following steps are based on using a pre-configured protocol with the demo dataset. You may also use your own images and settings as needed.
6. Select the Reset drop down and press the Load Demo button. The Open Demo Image option should also be checked to load the demo image at the same time.
7. Press the Sync button to load the active image and select the image or image channel containing the image to define the central core from the “Setup” combo box.
   The demonstration contains 2 layers around the central core and may be modified for different samples.  Set the “Outer Layer” to the Mono image from the combo box and the “Inner Layer” to the Mono image from the combo box.
   Note: After the central core is defined in the protocol, the protocol is designed to define the outer most layer first and then work inward towards the central core through the various inner layers.
8. By default, the Spatial and Intensity selections are set to “Current” and will use the image spatial and intensity calibrations associated with the image if they exist, otherwise the images will be uncalibrated. 
   If an image(s) is uncalibrated and calibrated values are desired, a calibration may be created using the Calibration group located in the Home or Capture tab before running the protocol.  Once an appropriate calibration(s) have been created, they may be selected using the appropriate calibration combo box.
9. Turn on Interactive and Press the Next Step button.

Concentric Layer Protocol Center Core Magic Wand Segmentation: Step 2

For more information on segmentation, please refer to the Protocols Help section: Protocol Segmentation.

10. Magic Wand Method: by default, the sample core area is segmented using the Magic Wand tool.  Click in the core area and the area will be segmented. The alternative method is to use the Manual Tag method and is described in the section below.
    1. Center Definition: Select method to define the core area center using the combo box.
    1. by Magic Wand: segmentation tool used to define the core area center.  The magic wand method is the default method.
    2. by Manual Tag:  tool used to click in the center of the core area.  Left mouse click in the image to set the center of core area.
    2. Sampling Frequency: number of measurement lines used to measure the thickness of the layers.  The lines are displayed the core area segmentation is completed.
    3. Show Measurement Pattern: The pattern will be automatically turned on after the center is define by either the Magic Wand or Manual Tag method.
    4. Flip analysis direction: turn on to inverse direction of the analysis.

11. If the segmentation of the core area needs adjustment, use a combination of the Smoothing and Histogram tools improve the segmentation of the core area.  Once the segmentation of the core area is correct, press the green Validate button to apply the changes.
12. Press the OK button in the Interaction dialog box.
    The sampling pattern used to measure the layer thickness is now displayed on the image.
13. Adjust the Sampling Frequency spin box to change the number of sampling lines if needed.
14. Press the Next Step button to continue.

Concentric Layer Protocol Center Core Manual Tag Segmentation: Step 2

15. Manual Tag Method: the sample core area center is defined by selecting the Manual Tag method in the Center Definition combo box.  Left mouse click in the center of the core area to define the point where sampling lines will terminate.
    If the center is incorrect, press the Define Center button and click in the center of the core area
    Adjust the Sampling Frequency spin box to change the number of sampling lines if required.
16. Press the Next Step button to continue.

Concentric Layer Protocol Outer Layer Segmentation: Step 3

17. Each layer is defined by a top and bottom boundary type.  They boundary type may consists of the following options, which allows the flexibility to either use segmentation or manual drawing methods to define the top and bottom boundaries of the layer.
    Select the polygon top, polygon bottom or polyline to define the top and bottom boundary layer for the outer layer.
    5. Top and Bottom Boundary Layer: use the combo box to select how the top and bottom layer will be defined.
    1. Polygon Top: Magic Wand, Manual Polygon drawing, or Polygon Auto-trace is used to define the polygon.  The top of the resulting polygon is used to define the top of the outmost layer in this protocol
    2. Polygon Bottom: Magic Wand, Manual Polygon drawing, or Polygon Auto-trace is used to define the polygon.  The bottom of the resulting polygon is used to define the bottom of the outmost layer in this protocol.
    3. Polyline: a manually drawn polyline is used to define the boundary layer.

18. Select Magic Wand or Manual to define a polygon boundary or the Manual polyline tool to manually draw the boundaries or boundary based on the how the layer boundary is defined in this step. This step in the protocol uses the Magic Wand to create the polygon around the outer layer.
    6. Magic Wand: is the default method used to define a polygon around the layer area.
    1. Histogram: adjust the sliders to change the segmentation range.
    2. Smoothing: low pass filter used to blur image to help identify layer boundary by magic wand.  Only the LUT is changed and not the image intensity values.
    7. Manual: 
    1. Select: activates the Select tool.
    2. Polyline: activates the Polyline drawing tool.  Use the dropdown to select Auto Trace Polyline.
    3. Polygon: activates the Polygon drawing tool.  Use the dropdown to select Auto Trace Polygon.
    4. Split Tool: activates the manual split tool.  Draw splitting lines on the image and click Apply Split button.  Click the button again to cancel the split operation.
    5. Delete: deletes and the selected segmented area on the image. To delete an object, press the Select button and click on the individual object.  ress the Red X Delete button to remove the object.

19. Press the Apply button to save the changes to the protocol if the button is active.
20. Set the Minimum Area and Fill Holes options as need to define the core center.
    Min Area: Sets the minimum object size to be included in the analysis.  The Edit Range button is used to set the minimum and maximum object size using the interactive histogram to set the limits.
    Fill Holes: turn on the option to fill holes within the layer.
21. Press the Apply button to save the changes to the protocol if the button is active.
22. For the Concentric Layer protocol, the Interactive Step is on by default.  The underlying assumption is central core area layers will need to be manually defined.
23. Press the Next Step button or press the Back button if the segmentation or other steps in the protocol need further adjustment.

Concentric Layer Protocol Inner Layer Segmentation: Step 4

In Step 4, the inner layer is defined using the same method and tools as described in the previous Step 3.  Notice in the settings, a new option is present to define the Top Boundary Type.  In this case, the Previous Layer may be used to define the Top Boundary Type of the inner or next layer since the division between the two layers has already been defined by the outer layer. 
The Bottom Boundary of the inner lay may be defined using a polygon or polyline tool.  Since the layers in the sample are well defined, the Magic Wand is used to create the polygon to segment the inner layer.

Create the polygon inner layer boundary using the Magic Wand and press the OK button in the Interaction dialog box to continue.

Concentric Layers Protocol Results

For more information on configuring and showing analysis results or saving protocols, please refer to the Protocols Help section: Configuring Results.

Once the resulting data has been configured and displayed, additional images may be analyzed individually or in a batch mode.