Confluence Protocol

Introduction

The Confluence protocol provides a simple workflow to measure the percentage of the available area occupied by cells in the image. In addition to measuring confluence as a percentage, the protocol reports an estimated cell count for the imaged area.

Protocol Information: 

  • Inputs: One brightfield or fluorescent channel
  • Time: End Point or time-lapse. 
  • Data Output: 
    • Total Cell Count, Estimated Cell Count, Total Cell area, Average Cell Area, Average Cell Diameter and Average Cell Intensity.
    • Optional snapshots of the analyzed locations with overlays.

Confluence 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 Confluence 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 Confluence protocol located in the Cell Biology 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 channels, if your dataset contains multiple channels, select the channel that contains images of cells from the “Cells” combo box. 
  7. Select the Interactive Mode check box and Press the Next Step button.

Confluence Protocol Segmentation: Step 2

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

  1. Segmentation

    1. Cells are automatically segmented using the default or saved protocol parameters.

    2. The confluence protocol includes 4parameters which may be adjusted individuallywith spin buttons or the slider bar.  The slider bar is displayed when hovering the mouse pointer over one of the controls as shown below.

      Threshold (%): Segmentation threshold as a percentage of the default value.  Decrease the value todetect more cell areas.

      Smoothing: Size of a Low Pass filter used to remove noise and blur cellsfor improved segmentation.

      Cluster Size (pix): Approximate size of cell clusters, used in setting a background correction filter for improved cell segmentation.  Higher values will increase the sensitivity of the confluence measurements.

      Close Filter (pix): Morphological image processing filter used to close gaps inside cells or between confluent cell areas. Increase the value to increase the fill area.

      Continue to adjust the parameters until the cells are properly segmented.

  2. The Segmentation Options may be used to further refine the identified cell area. Adjust the segmentation options as needed.  Press the Apply button to apply the changes to the protocol.

    1. Minimum Area: press the “Edit Range” button and set the minimum and maximum limits of valid cells. Objects outside of these limits are ignored by the protocol.    Press the OK button when done. 

      Note: Protocols may contain predefined settings and may require adjustment for proper cell identification.

    1. Clean Borders: removes cells touching the edges of images if selected.

    2. The Reference Size is the size of a typical cell, from which the number of cells in segmented areas can be calculated.  To set the reference size select a single segmented area that contains a known number of cells. 

      1. Press the Select button.

      2. Select a single cell or cell cluster.  Press the OK button when done.

      3. Enter in the number of cells contained within the selected area. Press the OK button when done.

  3. Press the Next Step button or press the Back button if the segmentation or other steps in the protocol need further adjustment.

Confluence Protocol: Results

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