After imaging the drug-treated C.elegane, open the ND file in the image J server with the colocalization plugin and select the split images option in the dialogue box. Each ND file contains image planes taken at three wavelengths and visible light. Work with brightfield, green, and red images.
Generate duplicates of these images to keep the original image untouched by clicking on image and selecting duplicate or using the keyboard shortcut shift plus D.Then reduce the background by generating another image duplicate as demonstrated earlier. Subtract the background with a rolling radius of 100 and select the create background option to generate an image with the given image's Background. Now go to process, click image calculator and subtract the first duplicated image from the second duplicated image.
Use the resulting images for the colocalization analysis. To use the colocalization plugin, convert the green and red channel images to eight bit by clicking image, selecting type, followed by eight bit. Next, click on plugins and select colocalization.
To measure the colocalization of mitochondria and lysosome signals, set the ratio to 75%the threshold red channel to 80.0, and the threshold green channel to 50.0. An eight bit binary image containing colocalized puncta and combining the three eight bit images in an RGB image is generated as output. Next, focus on the puncta in the head body wall muscle of the worms by manually selecting the area and creating a mask by clicking edit, selection, and create mask to select the region of interest.
To select the particles in the region of interest, use the image calculator to select the colocalized eight bit and masked images. Utilize the operation and to select the puncta in the region of interest generating an image with puncta in the region of interest. Finally, to analyze the area of the colocalized mitochondria and lysosomes, select analyze, followed by analyze particles and measure the summation of the puncta between 0.1625 square micrometers and four square micrometers.
The confocal images of the head body wall muscles of worms showed colocalization of mitochondria and lysosomes. VL-850 induced robust mitophagy in the worm's head body wall muscles, indicating a potent mitophagy inducer. The mitophagy potency of VL-850 was similar to that of FCCP.
