Optical Flow (Motility)

Configuration of the GUI

Motility in wrmXpress is measured using Farneback's dense optical flow algorithm implemented in OpenCV. This is in contrast to other published tools for measuring worm motility, which use the Lucas-Kanade algorithm that focuses on keypoints. Motility can run on data in the ImageXpress file structure or the AVI file structure (see Expected input below).

In Pipeline Selection, choose Optical Flow. The OpenCV function includes six parameters that can be adjusted. The following is taken directly from the OpenCV docs:

1. pyrScale (Default = 0.5): The image scale (<1) to build pyramids for each image; pyr_scale=0.5 means a classical pyramid, where each next layer is twice smaller than the previous one. Past wrmXpress users have used 0.9 for adult Brugia and Schistosoma and 0.5 for all other worms.
2. levels (Default = 3): The number of pyramid layers including the initial image; levels=1 means that no extra layers are created and only the original images are used. Past wrmXpress users have used 10 for adult Brugia and Schistosoma and 3 for all other worms.
3. winsize (Default = 20): The averaging window size; larger values increase the algorithm robustness to image noise and give more chances for fast motion detection, but yield more blurred motion field. Past wrmXpress users have had success using values between 2 and 30.
4. iterations (Default = 7): The number of iterations the algorithm does at each pyramid level. Past users have used 5 and 7.
5. poly_n (Default = 5): The size of the pixel neighborhood used to find polynomial expansion in each pixel; larger values mean that the image will be approximated with smoother surfaces, yielding more robust algorithm and more blurred motion field, typically poly_n = 5 or 7.
6. poly_sigma (Default = 1.1): The standard deviation of the Gaussian that is used to smooth derivatives used as a basis for the polynomial expansion; for poly_n = 5, you can set poly_sigma = 1.1, for poly_n = 7, a good value would be poly_sigma = 1.5.

Expected input

Motility data may be analyzed in the form of individual TIF images per frame (i.e., the TimePoint structure utilized by ImageXpress) or in the form of raw, uncompressed AVI video containers. See the Data Organization page for more details. All experiments should include a single wavelength.

File structure: ImageXpress + Single Well

In the case of individual TIF images per frame, the directory structure should look like this:

In Instrument Settings, choose ImageXpress + Single Well as the File Structure. Motility has not been tested with a Multi Well or Multi Site ImageXpress format.

File structure: AVI

In the case of AVIs, the directory structure should look like this:

If the AVI includes multiple wells per video, then the plate directory should include a single AVI with the same plate name.

In Instrument Settings, choose AVI + Single Well or AVI + Multi Well as the File Structure.

Validated species and stages

Filarial nematodes (i.e., Brugia malayi and Dirofilaria immitis)

• Microfiliariae
• L3s
• Adults

Schistosomes

• Adults
• Schistosomula
• Cercaria
• Miracidia (but we recommend using the Tracking pipeline instead)

Caenorhabditis elegans

• Larvae
• Young adults
• Gravid adults

Example plates

• 20210819-p01-NJW_753: Brugia malayi microfilariae, 96-well plate, individual wells (ImageXpress + Single Well)
• 20220622-p02-KTR: Brugia pahangi adults, 96-well plate, whole plate (AVI + Multi Well)
• 20220527-p02-KTR: Brugia pahangi adults, 24-well plate, whole plate (AVI + Multi Well)

Expected output

A CSV file with at least two columns: Well and Total Motility. Variation in the number of worms per well can be corrected for by dividing the motility value by the area value, which can be calculated with the Segementation pipeline. If using Metadata, there will be an additional column for each provided metadata CSV.