Hello Everyone! I'm posting my protocol for mounting and imaging worms.

Method for Mounting Worms and Imaging

Mounting

Start by leaving the agar in high heat to melt it, and switch to low heat once it is liquefies

Create the anesthetics mix by taking 2 ul of 0.2% tricane (TR), 4 ul of 0.02% tetramisole, and 94 ul of M9 buffer and put into a seperate eppendorf tube for a total of 100 ul of anesthetics mix. Use the electronic pipette and adjust the amount that you will be extracting.

Once the agar is melted, take a droplet of it using a glass pipette and drop it onto a new glass slide. Use the glass slide with taped edges to softly flatten the agar drop, allowing it to solidify and create a thin surface. Slowly slide off the taped glass slide and leave the agar sheet on the new glass slide.

Drop 4-5 ul of the anesthetics mix onto the agar pad

Pick worms and transfer them to the anesthetic liquid on the agar pad

Once you are done transferring your worms, squeeze the vaseline tube and create a thin barrier around the circumference of the agar pad. Then, place a coverslip over both the agar pad and the vaseline barrier. Your slide is ready for imaging.

Imaging

Click on the program on the desktop called “Infinity Pro”

Turn on the microscope by clicking both the on switch on the front and on the right side

Place the slide onto the stage. Adjust to 4X magnification by using the coarse and fine focus, as well as using the proper lens and moving the stage, once you have focused on the worms switch the magnification to 10X.

Hit “preview” to start the camera.

Pull the entire rod located on the top right hand side of the microscope out until you reach the last stop to allow the camera view to be displayed on the computer screen. Now, you will not see anything by looking into the microscope, but you can control the magnification and positioning by using the coarse and fine focus on the microscope and viewing the slide on the computer screen.

By turning off the right switch, you can slide up and down the light exposure controls.

Before, you take a picture, make sure the window magnification is at 40% by looking at the bottom right of the screen.

Take a picture of your worms by clicking Capture, and adjust the magnification or stage to take more pictures.

Save your pictures by clicking File---> Save As----> Your Personal Folder. The folders should be labeled by the date you take the images.

Label them in this format: Strain_40%_bodylength10x_1.tif. The strain is the strain of worm you are looking at (tmr17, av740, MSC1, etc), 40% stands for the window magnification of the picture, bodylength stands for the fact that are imaging the entire body for the worm, 10x stands for the magnification, and “1” stands for the number of the picture you are taking if you take more than one picture of the same worm, it should be labeled the number of the picture and a period and another number ie. tmr17_40%_bodylength10X_1.1 and so on .

Take a picture of the micrometer in the same conditions as all of the other images you took: at 40% magnification and at the magnification the rest of the images are in. Save this picture in the same folder as your other images of that day. You will use this to calibrate the measuring tools in ImageJ to measure your worms.

Turn off the camera and microscope, and cover the microscope with the bag to avoid dust and damage. Dispose of your slide and put away the micrometer in the drawer.