RS-FISH is a fast spot detection method optimized for performance.

Pipeline usage

To use RS-FISH instead of AirLocalize, you must pass the --use_rsfish parameter to the pipeline, and then specify the RS-FISH parameters, e.g.

--use_rsfish --rsfish_min 0 --rsfish_max 4096 --rsfish_anisotropy 0.7 --rsfish_sigma 1.5

More information about the parameters, including controlling the Spark cluster size, is included in the parameter documentation.

Interactive Parameter Finding

You can use the RS-FISH Fiji Plugin to interactively find the correct parameters for your data as follows.

  1. Use File->Import->N5 to open your data set and select the “Crop” option to cut out a small but interesting region for testing spot detection.
  2. File->Save As->Tiff… and reopen the TIFF image.
  3. Install RS-FISH plugins: Help->Update… then click “Manage Update Sites” and enable “Radial Symmetry” then click Close and restart Fiji.
  4. Plugins -> RS-FISH -> Tools -> Calculate Anisotropy Coefficient. Save this coefficient for later use. It should stay consistent when using the same microscope.
  5. Plugins -> RS-FISH -> RS-FISH
    • Set the anisotropy coefficient from step 4
    • Click Ok and Done on the options dialog
    • Set the minimum value in the intensity distribution
    • Click “OK press to proceed to final results”
  6. RS-FISH -> Tools -> Show Detections in BigDataViewer

Iteratively adjust the parameters until you are detecting all the spots you want.

At the end, you can choose RS-FISH -> Advanced to view all of parameter values, or record to a macro to save them. This will give you all the parameters you need to run the pipeline:

run("RS-FISH", "image=cropped.tif mode=Advanced anisotropy=0.7213 robust_fitting=RANSAC compute_min/max use_anisotropy sigma=1.50000 threshold=0.00500 support=3 min_inlier_ratio=0.10 max_error=1.50 spot_intensity_threshold=269.54 background=[No background subtraction] background_subtraction_max_error=0.05 background_subtraction_min_inlier_ratio=0.10 results_file=[]");