Sample image and/or code
D69_OPAD_CFSE_aGc647_NE555_1006.tif (4.0 MB)
- Upload an original image file here directly or share via a link to a file-sharing site (such as Dropbox) – (make sure however that you are allowed to share the image data publicly under the conditions of this forum).
- Share a minimal working example of your macro code.
- What is the image about? Provide some background and/or a description of the image. Try to avoid field-specific “jargon”.
These are images of human cells (neutrophils) and bacteria (Neisseria gonorrhoeae). In these images, the following are immunofluorescently labeled:
- total bacteria (both inside and outside)- labeled with CFSE - appear green
- bacteria outside of the neutrophil - labeled with an antibody - appear pink
- neutrophil elastase, a protein in the neutrophils - labeled with an antibody - appears red
In some images, the green bacteria have red neutrophil elastase staining around them, and we call this “enrichment”. When the area around a bacterium is enriched, the red staining creates a halo that is at least 50% of the circumference, and it is tight to the bacteria (we sometimes compare it to looking like the bacterium is in a “coffee bean” of red staining)
- What information are you interested in getting from this image?
I want to preface this by saying I am very new to using Image J or any image analysis software - let alone using masks successfully - and I also have no experience coding for image analysis - so please keep that in mind when responding to my post.
Our main goal is to determine – for each intracellular bacterium (green, but not pink), is it enriched with neutrophil elastase (red)?
We would then like to enumerate how many are enriched (positive) and how many are not (negative), to get a proportion.
As far as I can tell, this involves at least doing the following:
• Determining which bacteria are in focus enough to analyze accurately
• Discriminating between intracellular and extracellular bacteria (using the extracellular stain, pink)
• Instructing the analysis tool to compare the red staining directly surrounding a bacteria to what is immediately surrounding that and determine if it is enriched
• Repeating this process for many bacteria and many cells over many images in a set.
- What stops you from proceeding?
- What have you tried already?
- Have you found any related forum topics? If so, cross-link them.
- What software packages and/or plugins have you tried?
- The primary challenge with these images is the high background of the red staining in the neutrophils - it is diffuse throughout the cytoplasm and membranes. Occasionally there will be spots or entire regions of high intensity and enrichment that aren’t near a bacterium. Therefore it really isn’t possible to use just the intensity of the red staining to determine if a phagosome is positive for red enrichment or not. It’s possible that for accurate analysis, some bacteria, despite meeting the criteria of being in focus and intracellular, will have to be excluded because they reside in neutrophils that contain red staining that is too intense throughout, making a determination of enrichment impossible.
- We have tried analyzing by hand- but we end up with vastly different results depending on which individual is analyzing. Therefore, we need an objective, unbiased analysis tool.