Cell Counting Protocol

Introduction

The Cell Counting protocol provides a simple counting workflow to count the population of fluorescently labeled cells.  Cells may be labeled with a nuclear dye or a whole cell dye. In addition to the cell count, intensity and morphometric parameters are also be reported.

Cell Counting Protocol Information: 

  • Inputs: One image channel of fluorescently labeled cells.
  • Time: End Point  
  • Data Output: 
    • Total Cell Count, Estimated Cell Count, Total Cell area, Average Cell Area, Average Cell Diameter and Average Cell Intensity.
    • Optional snapshot image of the analyzed location(s).

Cell Counting Protocol Setup: Step1

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 cell counting can be found in the File/Open/Demo Images in the Protocol Cell Count 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 Cell Count 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 “Cell” combo box. 
  7. Turn on Interactive Mode and Press the Next Step button.

Cell Counting Protocol Segmentation: Step 2

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

  1. Segmentation
    1. Adjust the nuclear or cell segmentation using the histogram slider or press the Auto-bright segmentation button to automatically segment the cells. 

    When using Auto-bright thresholding, the auto-thresholding may be fine-tuned with the Standard Deviation offset.  Use a lower value to broaden the segmentation range.

    Press the Interaction OK button when done adjusting the segmentation.

  1. If threshold-based segmentation is not effective with your data, the segmentation method may be changed to Smart Segmentation.
  2. Adjust the segmentation options as needed to identify the cells.  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 valid cells. 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. 
    4. Clean Borders: removes cells touching the edges of images if selected.
  1. Press the Next Step button or press the Back button if the segmentation or other steps in the protocol need further adjustment.

Cell Counting Protocol: Results

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