Fiber Thickness Protocol

Introduction

The Fiber Thickness protocol is designed to measure the thickness and orientation of fibers optical and electron microscopy images.

Fiber Thickness Protocol Information:

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

Fiber Thickness 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 fiber thickness analysis can be found in the File/Open/Demo Images in the Protocol Materials/Fiber Thickness folder. The following instructions are based on using the demonstration image.
  1. Select the Count/Size tab.
  2. Press the Protocols button to display the protocol browser.
  3. Load the default Fiber Thickness protocol located in the Materials collection
  4. 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.
  5. 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.
  6. Press the Sync button to load the active image and select the image or image channel containing the fibers from the “Fibers” combo box.
  7. 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.
  8. It may be required to use a Region of Interest (ROI) to define the area of the image to analyze, to avoid calibration bars on the image or limiting the analysis to a specific area or structure
    Press the     Create ROI button under the Region of Interest section.  Draw the ROI and press the OK button in the interactive dialog box when done.  The ROI may also be deleted by pressing the Double Red X button located next to the Create ROI button.

Note: The ROI will be applied to all images when analyzing images individually or in a batch processing mode.

  1. Turn on Interactive Mode and Press the Next Step button.

Fiber Thickness Protocol Segmentation: Step 2

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

  1. The Segmentation Options section contains three main controls used to segment the fibers: Threshold (%), Smoothing and Max fiber width. Using the sliders (however over the control area to see the slider control) or spin button controls, adjust the parameters to segment the fibers.  By default, the option to Show Preview Mask is on and adjustments to the settings will be immediately visualized.
    1. Threshold (%): adjust the intensity threshold by adjusting the slider or spin buttons. A lower value increases the sensitivity of the segmentation.
    2. Smoothing: low pass smoothing filter kernel size to blur noise and smooth edges/fibers.
    3. Max fiber width (pixels): approximately defines the maximum width of the fiber. The parameter is used in Hight Pass filter separating fibers from background. Larger values can be used on high contrast images, where background intensity is very different from fiber intensity.
    4. Press the OK button in the Interaction popup when done adjusting the segmentation.
  1. Additional segmentation options are used to adjust the segmentation settings based on fiber type.
    1. Dark Fibers: Turn on for measuring dark fibers on a bright background.
    2. Show Preview Mask: On by default to show a preview segmentation mask, which is used to visualize changes to the segmentation settings.
    3. Hollow Fiber: Turn on the option if the fibers are hollow and the intensity in the middle of the fiber is approximately the same as the background intensity.
    4. Custom Method: Turn on the option to use Smart Segmentation as the fiber segmentation method.  Press the Adjust button located in the Segmentation group to launch Smart Segmentation.
  1. Adjust the parameters in the Measurement Options section to set how the measurements will be made.
    1. Measure Type: select the combo box to choose the type of fiber thickness measurement.
    1. Equidistant: thickness is measured on fiber segments with the given interval defined by the Step parameter (pix). This mode should be used when Average fiber thickness should take into account length of every fiber (longer fibers will have bigger contribution to the Average value) or variations of the thickness along the fiber should be taken into account.
    2. Central Point: thickness is measured only in the central point of fiber segments. This mode should be used only when one measure per fiber segment is necessary.
    1. Step (pixels): – defines the interval between thickness measurement lines along the fiber in Equidistant mode.
    2. Min length (pixels): defines the minimum length of fiber segments to be measured in pixels. The parameter is also used to define offset from the crossing points of the skeleton to create thickness measures.
  1. Press the Apply button to save the changes to the protocol.
  2. The Manual Edit section is used to fine tune the analysis adding or deleting individual fiber thickness measurements on the images.
    1. To delete a fiber measurement, press the Select button and click on the individual fiber measurement.  Press the Red X delete button to remove the measurement.
    2. Additional measurements may be added by either method listed below.
    1. Press the Line button and draw new measurements on the image.
    2. Press the Snap Line button and draw new measurements on the images.
    • Select: activates the selection mode.
    • Line: activates Line tool to draw new thickness measurements manually.
    • Snap Line: activates Line tool with Auto Snap to fiber edges. The line should be drawn across the fiber and it will automatically be snapped to fiber edges when the mouse button is released.
    • Delete: deletes and the selected measurement on the image.
  1. Press the Next Step button or press the Back button if the segmentation or other steps in the protocol need further adjustment.

Fiber Thickness 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.