TITLE: C. elegans worm size measurement
AUTHORS: Laura Breimann

[DESCRIPTION]
Protocol to detect the size (length + width) of worms using a stereoscope and a camera.  


The protocol was used in the publication: 
 


[STEPS]
SECTION: Preparing worms for imaging
1. Select worm line(s) and prepare the worms for size measurement.
 

SECTION: Imaging worms
8. Use a microscope calibration ruler and take an image with the same settings. This will allow you later to give the results a unit.

  

Microscope calibration rulers can be found here.

SECTION: Imaging worms
7. To capture images of the worms, an eyepiece camera (for example the Dino-Eye camera) can be used. It is also possible to capture images using a phone with an adapter. Phone adapters can be 3D printed with many available designs online. Importantly, keep the zoom of the camera and the microscope constant for one experiment. Try not to image worms twice. If the camera allows it save raw file formats otherwise use the camera format that is available. 
 

SECTION: Preparing images for analysis
9. Save images for one condition in a folder. 
SECTION: Measuring worm size
13. Download the WormSizer plugin through the Fiji plugin menu. In Fiji navigate to "Help" > "Update" > "Manage Update Sites" and select "WormSizer" in the list of plugins and click "Close" and "Apply changes". After restarting Fiji, the plugin can be found in the Plugin drop-down menu. 
 


  

More info on how to use the plugin can be found here.

SECTION: Measuring worm size
14. Select an input directory including your pictures and an output directory to save the results. Click the >> button to continue. 


  


SECTION: Plotting results
20. The results file can be used to plot the results. An example script using Python can be found in this GitHub repository.


  
SECTION: Preparing worms for imaging
4. Wash worms off the plate using M9. To remove all bacteria, let worms settle in a tube for 10 min and carefully replace the M9 with fresh M9. Repeat this step 3 times. After the last step, remove most M9 from the tube, leaving only 20 µl with the worms. 
SECTION: Preparing worms for imaging
2. For the synchronizing-by-picking method, pick (at least) 20 gravid Adult worms and place them on a fresh OP50 plate. Allow worms to lay eggs for 4 hours and then remove Adults from plates.
 

SECTION: Preparing worms for imaging
3. Grow worms to the desired stage.
SECTION: Preparing worms for imaging
5. To get a clear picture of the worms, lightly anesthetize the worms by adding 10 mM Levamisol to the tube. Wait 5 minutes and gently pipette worms onto an unseeded agar plate. 
 

SECTION: Preparing worms for imaging
6. Separate the worms from each other using an eyelash pick. The goal is to have evenly spread worms that are not touching and are not too close to the wall. 
 

SECTION: Preparing images for analysis
10. Use Fiji to open the images and remove background. 
 
Open image using Fiji
Set the image to 8-bit: Image > Type > 8-bit
Subtract background: Process -> Subtract Background. Set the preview checkbox to see how changing rolling ball radius changes the results.
  
4. Threshold: Image -> Adjust -> Threshold; where it says “Default”, change it to “Otsu”. Move the sliding bars so that you get as much of the worms highlighted, and as little of the background. Then click “Apply”.

  


5. Save the image to a new folder for further analysis.  
SECTION: Preparing images for analysis
11. To run the above code on many images, you can use this Fiji macro. Make sure to adapt the parameters in the script after finding them manually. 

https://github.com/ercanlab/2021_Breimann_et_al/blob/main/WormSize_analysis/prep_worms_for_wormsizer.ijm
SECTION: Measuring worm size
15. Set the length in microns that were measured for the calibration ruler. Click the >> button to continue (this can take a moment depending on the number of images in the folder).
  

SECTION: Measuring worm size
16. Next, select the settings to find the worm outlines. Depending on the images and the magnification they will be different. After the selection, pressing the >> button will start the detection in all images in the folder. 
  
The processing complete image will signal when the plugin detected all worms and the review can start. 
  


SECTION: Measuring worm size
18. Reviewing the detections is done using the magnifying symbol. Navigate to the output folder and select the .xml file.  
  

SECTION: Measuring worm size
19. In the review part of the plugin, you can go through all detected worms in all images and select whether you want them to be included in the downstream analysis.

  
  

  
The results file is updated while reviewing and changes the value in the "pass" column to either TRUE or FALSE.
SECTION: Measuring worm size
17. The plugin produced three files in the output folder: 

settings .csv file
experiment .xml file
results csv file

The settings file saves the settings selected for detection. The XML file saves the outline information about each worm and can be used to review the outlines. The results file contains the worm measurements for each detected worm.  

  

SECTION: Preparing images for analysis
12. To get the resolution of your measurements, open the image of the calibration ruler. 
Use the line tool in Fiji 
  
Draw a line between two known points on your ruler (for instance a millimetre) 
Press command + M to open a results Window with the measurements to give you the length in pixels.
  
For “Microns per Pixel”, divide the number of microns your line is (In our case it was a mm, so it would be 1000 microns), by the length of the line in pixels from the step above.
Write down this number for the next step


Alternatively, read here for more instructions.