Cell Morphology Protocol

Introduction

The Cell Morphology protocol measures the morphology of fluorescently labelled cells containing fluorescently labelled nuclei.

Cell Morphology Protocol Information: 

  • Inputs: Multi-channel fluorescence image containing 1) a fluorescently labeled nuclei channel, and 2) a fluorescently labelled cytoplasm or whole cell channel.
  • Time: End Point  
  • Data Output: 
    • Per cell data: Area, Aspect Ratio, Axis Major,Axis Minor, Clumpiness, Diameter Mean, Fractal Dimension, Heterogeneity, Intensity Mean, Intensity Std Dev, Margination, Diameter, Equivalent Circle, Perimeter Ratio, Perimeter Bounding Pixels, Radius Ratio, Roundness, Volume Equivalent Sphere
    • Statistics: Area(Mean), Area(St Dev), Aspect Ratio(Mean), Aspect Ratio(St Dev), Axis Major(Mean), Axis Minor(Mean), Clumpiness (Mean), Clumpiness(St Dev), Diameter, Equivalent Circle(Mean), Diameter Mean(Mean), Diameter Mean(St Dev), Fractal Dimension(Mean), Heterogeneity(Mean), Heterogeneity(St Dev), Intensity, Mean(Mean), Intensity, Mean(St Dev), Margination(Mean), Margination(St Dev), Perimeter Ratio(Mean), Perimeter Bounding Pixels(Mean), Radius Ratio(Mean), Radius Ratio(St Dev), Roundness(Mean), Roundness(St Dev), Volume Equivalent Sphere(Mean),Volume, Equivalent Sphere(St Dev)
    • Optional snapshot image of the analyzed location(s).

Cell Morphology 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 cell morphology analysis can be found in the File/Open/Demo Images in the Protocol Cell Morphology folder.  The following instructions are based on using the demonstration image.

  2. Select the Count/Size tab.

    Press the Protocols button to display the protocol browser.   

  3. Load the default Cell Morphology 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.  Select the appropriate channels in the ‘Nucleus’, and ‘Cells’ combo boxes. 

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

Cell Morphology Protocol Nuclei 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 segmentation using Smart Segmentation. Press the OK button in the Interaction popup when done adjusting the segmentation.

  2. The segmentation method may also be changed in the protocol by selecting Threshold Segmentation and completing the segmentation step.

  3. Adjust the segmentation options as needed to identify nuclei.  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.

      Note: Some protocols contain predefined settings and may need to be adjusted to suit your data.

    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.

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

Cell Morphology Protocol Cell Segmentation: Step 3

  1. Segment the cell channel using the histogram, auto bright or Smart Segmentation method as described in the previous section. Smart Segmentation is the default method.

    Press the OK button in the Interaction dialog box when finished adjusting the segmentation.

  2. Press the Next Step button when the segmentation step is complete.

Cell Morphology 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.