Neurite Outgrowth Protocol

Introduction

Measure fluorescently labelled neurons and cell bodies containing fluorescently labelled nuclei.

Neurite Outgrowth Protocol Information:

• Inputs: Multi-channel fluorescence image containing 1) a fluorescently labeled nuclei channel, and 2) a fluorescently labelled neurite channel.
  
• Time: End Point 
• Data Output: 
  
  • Per cell,well, or image: Nuclei Count, Total Neurite Length, and End-point Count
  • Optional snapshot image of the analyzed location(s).

Neurite Outgrowth 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 neurite outgrowth analysis can be found in the File/Open/Demo Images in the Protocol Neurite Outgrowth folder.  The following instructions are based on using the demonstration image.

2. Select the Count/Size tab. 

3. Press the Protocols button to display the protocol browser.   

4. Press the Cell Biology Advanced button on the left side of the dialog box.
   

5. Load the default Neurite Outgrowth protocol. 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

6. The demonstration image is a multichannel set. If using your own images and they are in a multiwell plate format, select a test well using the Plate Viewer.
   

7. From the Protocol dialog box, 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.
   
8. Press the Sync button to load the active image channels, if your dataset contains multiple channels, select the channel that contains the nuclei from the “Nuclei” combo box, and the neurites channel from the “Neurites” combo box.
   The channels should be set for the demo image as:
   Nuclei: DAPI channel
   Neurites: GFP channel
   
9. Turn on Interactive Mode and press the Next Step button

Neurite Outgrowth Protocol Nuclei Segmentation: Step 2

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

10. Segmentation
    1. Adjust the nuclear 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 located in the bottom right-side corner of the dialog box.  The Standard Deviation offset will help correct for variable intensity levels between wells or positions.
    

    Press the OK button in the Set Thresholds popup when done adjusting the segmentation.
    

11. If threshold-based segmentation is not effective with your data, the segmentation method may be changed to Smart Segmentation.
    

12. Adjust the segmentation options as needed to identify the cell bodies as described in the previous step.
    1. Minimum Area: press the “Edit Range” button and set the minimum and maximum limits of valid nuclei. 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.

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

Neurite Outgrowth Protocol Cell Body Segmentation: Step 3

14. Segment cell bodies using the Threshold (%) slider. Set the minimum cell body size using the Cell Body Size (pix) slider. With the Show Preview Mask option on, the segmentation mask will be updated as you adjust these parameters.  Cell bodies are divided based on the positions of nuclei in the previous step (so that each cell body contains a single nucleus), and objects that lack are nucleus are discarded.
    
    Threshold (%): adjust the intensity threshold by adjusting the slider or spin buttons. A lower value increases the sensitivity of the segmentation.
    
    Cell Body Size (pix): cell bodies smaller than the area in pixels will be discarded
    
    Press the OK button in the Interaction dialog box when done adjusting the segmentation. The cell bodies will be outlined.
    
    
15. Adjust the segmentation options as needed to identify the cell bodies as described in the previous step.
    1. Minimum Area: press the “Edit Range” button and set the minimum and maximum limits of valid nuclei. 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.

16. Press the Apply button to save the changes to the protocol.
    

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

Neurite Outgrowth Protocol Target Segmentation: Step 4

17. Segment neurites using a combination of: Threshold (%), Smoothing, Brach Width (pix) and Close Filter (pix) parameters. 
    
    Threshold (%): adjust the intensity threshold using the slider or spin buttons.  A lower value increases the segmentation sensitivity.
    
    Smoothing: a low-pass filter that blurs out background fluorescence and smooths (averages) the fluorescent signal to make objects solid.
    
    Neurite Width (pix): sets the minimum width or thickness of neurites.
    
    Close Filter (pix): The kernel size of a morphological close filter which closes gaps between neurites or within cells.   The segmentation options in this step include an additional method to close gaps between neurites.
    
    Press the OK button in the Set Thresholds dialog box when done adjusting the segmentation.
    

18. The cell bodies and neurites are displayed with the following overlays:
    
    1. Dashed Yellow polygon: cell body.
       
    2. Blue line: neurite “branch” terminating in an end point.
       
    3. Red diamond: node. Nodes are neurite branch points. The cell bodies are classed as “Nodes” when connected to at least one neurite.
       
    4. Green circle: neurite “branch” endpoint.

19. Once neurite segmentation is complete, additional options are available to further refine the neurite identification.
    Use the Segmentation Options drop down selector to view the options.  The Link and Prune options may be used in the following manner:
    
    1. Link:  connect gaps between neurite segments due to weak signal or spatial location.
       
    2. Prune:  remove neurites shorter than the value set.  Decrease this setting to include short segments which need to be connected to an adjacent neurite. Commonly, the Link and Prune options are used together to properly identify neurites in the image.
       
       For example, a smallneuritemay be removed because it was smaller than the Prune size.  By adjusting the Prune option smaller, additional small sections of neurites will now displayed and measured, once the Apply button is pressed.
       
       To close a gap, adjust the Link value larger and press the Apply button to see the results. 
    3. Press the Next Step button or press the Back button if the segmentation or other steps in the protocol need further adjustment.
       
       

Neurite Outgrowth 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.