Ring: 1 Ring, 1 Target Protocol

Introduction

This Protocol is designed to create a ring-shaped mask around the nucleus, cell, or other structures.  Within the ring mask area, the intensity information of a fluorescent targetis measured and reported.  The ring may be located outside, inside or overlapping the segmented area.

1 Ring, 1 Target Protocol Information:

  • Inputs: Multi-channel fluorescent image containing 1) nuclear or other labeled structure of interest, which is used to define the ring mask and 2) a separate fluorescentimage of the target.
  • Time: End Point 
  • Data Output: 
    • Per Ring: Ring Area, TargetMean Intensity and Target Count
    • Statistics: Number of Cells, Number of Target Objects, Area Sum of Target, and Target Mean Intensity.
    • Optional snapshot image of the analyzed location(s).

1 Ring, 1 Target Analysis 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 the protcol can be found in the File/Open/Demo Images in the Protocol Cell Biology/1 Ring 1 Target 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.
  1. Press the Cell Biology button on the left of the dialog box.
  1. Load the default Ring: 1 Ring, 1 Target protocol.
  2. 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.
  3. 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.
  4. Press the Sync button to load the active image channels, if your dataset contains multiple channels, select the channel that contains images of nuclei or cells from the “Parent” combo box and the target from the “Target” combo box.
    The channels should be set for the demo image as:
    Parent: DAPI channel
    Target: GRP channel
  5. Turn on Interactive Mode and press the Next Step button.

1 Ring, 1 Target Analysis 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 structure segmentation using the histogram slider or press the Auto-bright segmentation button to automatically segment the nuclei. 

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

  1. The segmentation method may also be used in the protocol by selecting Smart Segmentation and completing the segmentation step.

Ring Adjustment and Creation

  1. By default, the initial ring is set to grow 15 pixels out from the edge of the segmented structure, creating a 15-pixel thick ring mask area.
  2. The ring area may be changed by applying positive or negative values to the Grow controls.  For example, to create a ring area 30 pixels thick, with the outer ring boundary 20 pixels outside, and the inner ring boundary 10 pixels inside the segmented nuclei, apply the following settings:

Grow Size: 20
Ring Offset: -10

Press the Apply button.

  1.  Adjust the segmentation options as needed to identify the nuclei or structures.  Press the Apply button to save the changes to the protocol
    1. MinimumArea: 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.

    1. FillHoles: when selected, any holes within your objects will be filled.
    2. Split: select either Boundary Shape or the Watershed nuclei splitting methods to separate joined nuclei or None.
    3. CleanBoarders: removes nuclei touching the edges of images if selected.
  1. Press the Back button if the segmentation or other steps in the protocol needs further adjustment.
  2. Press the Next Step button.

1 Ring, 1 Target Analysis Protocol Target Segmentation: Step 3

  1. Segment the target channel image using the histogram, auto bright or Smart Segmentation method as described in the previous section.
    Press the OK button when done.
  2. Adjust the segmentation options if required.  If the default settings are changed, press the Apply button.
  3. Press the Next Step button.

1 Ring, 1 Target Analysis Protocol Results

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