Wound Healing Protocol

Introduction

The Wound Healing protocol measures the cell migration rate as the gap closes in scratch assays. The protocol is part of the ‘Cell Biology Advanced’ collection. The collection is an optional licensed Image-Pro module and will be available if purchased.

Wound Healing Information:

  • Inputs: Time resolved image set containing a bright field image sequence.
  • Time: Time lapse image series or sequence
  • Data Output: 
    • Wound Area Time Plot, Wound Area, Wound Width, Area Closure Rate, Edge Travel Rate, Time to 50% Wound Closure
    • Optional snapshot image of the analyzed location(s).

Wound Healing 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 wound healing analysis can be found in the File/Open/Demo Images in the Protocol Wound Healing 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 Wound Healing protocol located in the Cell Biology Advanced collection.
  4. The following steps are based on using the using a pre-configured protocol for 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 open at the same time.
  6. Press the Sync button to load the active image channel.  The Wound Healing protocol is designed to work with a set of time resolved images or a single image sequence file.  If the image sequence file is an RGB color image, please set the scratch channel to Color.
  7. Select the Interactive Mode check box and Press the Next Step button.

Wound Healing Segmentation: Step 2

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

  1. Segmentation
    1. Segment the scratch area in the image using the histogram, auto bright or Smart Segmentation method.  Smart Segmentation is the default method.
      Additional Reference and Background objects may be drawn to refine the smart segmentation if required.
      If additional reference objects do not lead to satisfactory results, press the Recipe Options Clear button. New reference objects and Background areas may be created on the image for creating a new recipe.
    2. Confirm or adjust the segmentation as required for additional time points by adjusting the sequence control toolbar slider.
      Press the Interaction OK button when done.
    3. The segmentation method may also be changed in the protocol by selecting Threshold Segmentation and completing the segmentation step.
  1. Adjust the segmentation options, if needed, to identify the scratch area.  Press the Apply button to save the changes to the protocol.
    1. Minimum Area: press the “Edit Range” button and set the minimum and maximum limits of the scratch area. Objects outside of these limits are ignored by the protocol.    Press the OK button when done. 
    2. Fill Holes: when selected, any holes within your objects will be filled.
    3. Split: select either Boundary Shape or the Watershed cell splitting methods to separate joined cells or None.  The selection is set to None for the Wound Healing Protocol.
    4. Clean Borders: removes cells touching the edges of images. This should be set to None for the wound healing protocol.
  1. Press the Next Step button or press the Back button if the segmentation or other steps in the protocol need further adjustment.

Wound Healing Protocol: Results

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

  1. The segmentation is completed and displayed on your setup image. If you are happy with the segmentation, you are ready to run the protocol on the full time series (steps 11-13).  Press the Back button if you want to make further adjustments to the protocol setup.
  2. Press the Finish button. When the Finish button is pressed, the segmentation overlay will no longer be displayed.
  3. Turn off Interactive Mode.
  4. Press the Run Selected button.

Note: All active well positions will be analyzed when the Run Selected button is pressed.  If you would like to check the segmentation protocol settings on additional wells, leave the Interactive Mode set to on and activate a different well.

  1. The wound analysis results will automatically be displayed when the analysis is completed.