Using WormSizer to measure Worm Length or Volume (PC) Edit

  • Install Leica Application Suite V4.5.0 from DVD

Place worms on unseeded NGM plate forimaging Edit

  • wash 20-50 worms 2-3x with M9 in a 1.7ml Eppendorf spinning in benchtopcentrifuge
  • place 20-50 individuals on unseeded NGM plate
  • spread out individuals for better images

Acquire images usingLeica M80 camera and LAS v4.5 for PC (since Fiji cannot handle acquisition) Edit

  • Setup Arrow: Make sure 0.5x or 1.25x
  • Options->Preferences->Image Tab->Select JPEG
  • Acquire Arrow, M80 Tab: select appropriate zoom to match the scope (4.0x)
  • Acquire Arrow, Camera tab: Automatic Exposure on
  • Hit Acquire Image, and save so that file name lists genotype, condition, etc.
  • The image #s should automatically increase
  • Browse Arrow -> button with “i” -> scroll to find “XMetersPerPixel” & save this number

Batch Convert images to 8-bit grayscale (since Leica cannot acquire 8bitGray, we are told) Edit

  • Open one of the images in Irfan View
  • Press “b” to open batch tool
  • Press “Add All” to add all images to list
  • The following only needs to be set up once, then just change the directory as needed
  • Select “Batch Conversion-Rename result files” and “JPEG”
  • Advanced: Change color depths/256Colors 8BPP/Convert to Grayscale
  • Hit OK, and Start Batch. You can verify bit depth in Properties - right click the image

Start Fiji Edit

  • Plugins -> WormSizer -> WormSizer GUI
  • Play button, then select desired folder of images for both input and output folder options
  • Input microns per pixel (the # saved from Leica software)
  • Hit >> and wait (if >1minute per image, and keeps “thinning”, it probably encountered a problem)
  • Magnifying glass button, then select desired .xml file
  • press P(ass) or F(ail) as appropriate

Find Fiji’s results.csv and open in excel Edit

  • select all and filter. Pass = TRUE
  • copy PASSed data to second sheet/workbook
  • calculate COUNT, AVERAGE, and STDEV in excel, or process in R
  • length:meanWidth may be useful as a kind of “BMI” for worms
  • Output volume is in picoliters, length & width are in microns