TITLE: Image processing to investigate NEMO recruitment and involvement in mitophagy and inflammatory signaling
AUTHORS: OLIVIA HARDING, Erika L.F. Holzbaur

[DESCRIPTION]
Beautiful images are not sufficient to robustly characterize and interrogate a cellular mechanism. In order to show recruitment of NEMO and its relationship to OPTN and other mitophagy factors, we processed hundreds of still and timelapse images and extracted quantifiable data in order to perform statistical analysis comparing different conditions. In many cases, we used software to deconvolve confocal fluorescent images, allowing us to algorithmically surpass the resolution limit. This was especially useful for live cell images that had been collected with low power settings to preserve cell health. We also employed machine learning software to generate binary segmentations and carry out particle analysis on putatively overlapping structures. Finally, in some cases we simply identified fluorescent structures by hand and measured their fluorescent intensities. We approached image analysis in a multitude of creative, effective ways, and importantly we maintained consistency of analysis within experiments in order to present the results with integrity and reproducibility. 

[BEFORE_START]

For each experiment, collect images for at least 10 cells from at least three biological replicates.
Maintain consistent imaging parameters by saving and/or recording laser powers and exposure times.

[GUIDELINES]
This protocol addresses multiple image analysis pipelines used in the corresponding manuscript to investigate NEMO and its role in mitophagy.
We developed several methods for quantitative analysis with our varying experiments.

[STEPS]
SECTION: Blinded whole-cell recruitment assessment
1. 
 

 Start with Z stack images of live cells collected with a 60X objective.
SECTION: Blinded whole-cell recruitment assessment
2. Max project the Z stack fields of view by ~2 um to capture the center to upper half of the cell.
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3.  Crop each cell and save only the NEMO channel with a file name to identify the conditions.
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4. Copy cropped NEMO channel images to a new folder.
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5. Use a random number generator such as Random.org/strings/ to generate a number of strings equivalent to the total number of cells.
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6. Copy the strings to a document and save.
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7. Rename the copied images in the new folder as the random strings.
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8. Arrange files in the folder by file name in order to randomly mix images.

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9. Open newly named files and judge whether there is NEMO recruitment or not, recording YES or NO for each image by its random string file name.
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10. After categorizing every image, unblind the results and determine the number of cells marked YES for each condition compared to the total for that condition.

SECTION: Mitochondrial recruitment assay
11. 
 

 Start with Z stack images of cells collected with 60X objective. 
SECTION: Mitochondrial recruitment assay
12. Crop and save each cell visible in the field of view.
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13. Deconvolve images with Huygens or similar deconvolution software.
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14. Max project each cell ~2 um.
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15.  Split channels and save each channel.
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16.  Import 5-7 channels displaying mitochondria into the Ilastik software for segmentation.
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17. Train algorithm to recognize “mitochondria” or “not mitochondria”.
 

SECTION: Mitochondrial recruitment assay
18. Generate binary images for all mitochondria channels.
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19. Repeat previous 3 steps for NEMO puncta and OPTN rings/puncta.
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18.1. Import segmented Ilastik results to FIJI.
 
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18.2. Use the original image file to draw an ROI outlining the cell.
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18.4. Run the threshold function with threshold set to (255,255).
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18.3. Clear Outside the cell ROI on the segmented image.
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18.5.  Save binary image.
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20. GABARAPs and LC3B antibodies do not produce high enough signal-to-noise to segment with Ilastik. For these experiments, only segment NEMO and mitochondria. In FIJI, draw ROI’s around GABARAPs- or LC3B-positive mitochondria by hand. Save ROIs.
SECTION: Mitochondrial recruitment assay
21. To determine the % of mitochondria that recruited NEMO,
 
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21.1. Open the binary mitochondria image in FIJI.
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21.2. Use the Analyze Particles function to generate a mask of binary image, filtering particles to exclude those fewer than 5 pixels.
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21.3. Summarize and add to manager.
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21.4. Record the total number of mitochondria in Excel.
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21.5. Open binary image of NEMO channel.
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21.6. Use the Analyze Particles function to generate a mask of binary image, filtering particles to exclude those fewer than 5 pixels, and add particles to manager.
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21.7. Project NEMO particles to Mito binary channel and multimeasure.
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21.8. Copy results to Excel.
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21.9. The count of particles with an average intensity of >127.5 is the number of mitochondria positive for NEMO.
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21.10. Divide this number by the total number of mitochondria to calculate the proportion.
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22. To determine the % of mitochondria that recruited NEMO and/or OPTN.
 

SECTION: Mitochondrial recruitment assay
22.1. Repeat the first four steps above to record total number of mitochondria.
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22.2.  Load the binary OPTN channel to FIJI and Fill Holes.
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22.3. Project mitochondrial particle ROIs onto OPTN binary image and multi-measure.
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22.4. Record results in Excel.
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22.5. The count of particles with an average intensity of >127.5 is the number of mitochondria positive for OPTN.
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22.6. Delete mitochondria particles from the manager and add NEMO particles.
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22.7. Project NEMO particles onto mitochondria and perform the same calculation.
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22.8. Use the image calculator to add the mitochondria channel to the OPTN channel with Filled Holes.
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23. To determine the % of mitochondria that recruited NEMO and/or GABARAPs or LC3B.
 
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22.11. Project NEMO particles to this new image and multi measure.

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22.10. Convert to an 8-bit image.
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22.9. Use Math > Subtract to subtract 255 and float the resulting image.
 
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22.13. The count of particles with an average intensity greater or equal to 64 is the count of mitochondria that are positive for both NEMO and OPTN.
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22.12. Record results in Excel.
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23.1. Use the Image Calculator to add NEMO particles to Mitochondria particles.
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23.2. Use Math > Subtract to subtract 255 and float the resulting image.
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23.3. Convert to an 8-bit image.
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23.4. Project hand-drawn ROIs to resulting image and multi measure.
 
SECTION: Mitochondrial recruitment assay
23.5. Record results in Excel.
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23.6. The count of particles with an average intensity greater or equal to 64 is the count of mitochondria that are positive for both NEMO and GABARAPs or LC3B.
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24. Use the R code library(eulerr) to generate Euler diagrams of the resulting data.
SECTION: Recruitment assay
25.  
 

 Start with confocal timelapse images in which the timepoint with added AntA/OligA is known.
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26. Choose several events per cell in which the mitochondria stays in the field of view for a majority of the timecourse and the protein of interest is recruited.

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27. For each time point, use the mitochondria channel to draw a generous ROI around the mitochondria.
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28.  Measure the intensity of NEMO and/or OPTN or p62 for every ROI and record in Excel.

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27.1. This will capture rings or puncta that are recruited.
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29. Calculate the 5-frame moving average across the timecourse for each channel.
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30. Calculate the background intensity by averaging the moving average intensity of the first ten frames of the timecourse.
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31. Subtract the background from every frame.
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32. Determine the maximum intensity for the event.
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33. Normalize the timecourse intensities to the max intensity so that the intensity of each frame is a percentage of the max intensity.
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34. Calculate the half-max timepoint based on when the normalized intensity surpasses 50%.