Colocalization Protocol

Introduction

Measure the correlation and overlap of two fluorescently labelled molecules.

Colocalization Protocol Information:

• Inputs: Multi-channel fluorescence image containing 1) a fluorescently labeled molecule channel, 2) a fluorescently labeled channel of a different type of molecule and 3) an optional fluorescently labeled parent object such as cells, nuclei, or mitochondria to measure colocalization within a specific structure.
• Time: End Point  
• Data Output: 
  • Per Parent (cell or structure) data: Area, Area for Coloc 1 and 2, Area of Coloc, Coloc Pearson, Pearson All, Overlap, K1, K2, M1t, M2t, Channel IOD, Channel Mean Intensity
  • Statistics: Total Objects, Population Counts, Area Sum, Area Sum for Coloc 1 and 2, IOD Mean Coloc 1 and 2, Intensity Mean Coloc 1 and 2
  • Optional snapshot image of the analyzed location(s).

Colocalization Protocol Setup: Step 1

For additional information on setting up protocols, please refer to the Help section: Protocol Setup. The following protocol example includes the option of having a “Parent” object such a cell, nucleus, or other structure.

1. Load your image dataset. An example of a dataset suitable for colocalization analysis can be found in the File/Open/Demo Images in the Protocol Colocalization 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. Load the default Colocalization protocol located in the Cell Biology Advanced collection.
5. 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. 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.
7. Press the Sync button to load the active image channels.  Select the appropriate channels in the ‘Parent’, ‘Coloc1’ and ‘Coloc2’ combo boxes. 
8. Select the Interactive Mode check box and Press the Next Step button.

Colocalization Protocol Parent Segmentation: Step 2

This an optional step in the Colocalization protocol.  If there is no Parent channel, the Set Colocalization Set Threshold step will appear.  For more information on segmentation and recipe creation, please refer to the Protocols Help section: Protocol Segmentation.

9. Segmentation
   1. Adjust the parent segmentation using the histogram slider or pressing the Auto-bright segmentation button to automatically segment the parent structures.  When using automatic bright thresholding, the auto-thresholding may be adjusted using the Standard Deviation offset (which is located to the right of the Auto-bright segmentation button).  The Standard Deviation offset will help correct for well-to-well intensity variation. Press the OK button in the Set Thresholds dialog box when done adjusting the segmentation.

10. The segmentation method may also be changed in the protocol by selecting Smart Segmentation and completing the segmentation step.
11. Adjust the segmentation options as needed to identify the parent structures.  Press the Apply button to save the changes to the protocol.
    1. Grow: grow or shrink the object outlines in pixels using the slider or spin buttons.
    2. Ring: create a ring shape outline where the analysis will be performed. 
    3. Offset: the option is active when the Ring is on.  Move the inner object boundaries in or out by adjusting the slider or spin buttons.
    4. Minimum Area: press the “Edit Range” button and set the minimum and maximum limits of valid parent objects. 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.

    5. Fill Holes: when selected, any holes within your objects will be filled.
    6. Split: select either Boundary Shape or the Watershed cell splitting methods to separate joined cells or None.
    7. Clean Borders: removes cells touching the edges of images.

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

Colocalization Protocol Set Colocalization Thresholds: Step 3

13. Set the minimum colocalization thresholds using the histogram slider with the channels locked together or independently adjusted by setting the lock button.  The Mode may also be changed from Threshold to Free ROI by selected the Mode drop down control.  The colocalization mask may be displayed or hidden to see the areas of colocalization on the image. Press the OK button in the Interaction dialog box when adjusting the threshold is completed.
14. Press the Next Step button when the colocalization threshold step is complete.

Colocalization 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.