Using LEICA scope and WormSizer (PC)

Using WormSizer to measure Worm Length or Volume (PC)

 * Install Leica Application Suite V4.5.0 from DVD


 * Download IrfanView from http://www.irfanview.com/
 * Download Fiji (ImageJ 1.48t) from http://fiji.sc/Downloads
 * Download and install WormSizer Plugin from https://github.com/bradtmoore/wormsizer

Place worms on unseeded NGM plate forimaging

 * 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)

 * 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)

 * 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

 * 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

 * 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