C. elegans Muscle Area Measurements: A Standardized Method to Quantify Striated Muscle Morphology

This protocol is excerpted from Teoh et al, Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans, J. Vis. Exp. (2019).

1. Quantifying body wall muscle structure

1. Fluorescence imaging of body wall muscle structure
   1. Obtain a strain (for example, RW1596) carrying the transgene stEx30(Pmyo-3::gfp::myo-3 + rol-6(su1006)), which labels myosin fibers with GFP.
      NOTE: Introduction of the transgene into animals can be achieved by either crossing a strain of interest with animals that already express the transgene, or microinjecting DNA into the distal arm of the gonad. The ‘rolling’ phenotype induced by rol-6 mutation in this transgene facilitates the visualization of the muscles. The body wall muscles run along the dorsal and ventral sides that are normally precluded from view in wild-type animals because they typically lie on one of their lateral sides. The rol-6(su1006) allele induces twisting of the animals, thereby exposing some muscles cells for imaging.
   2. Image the animals using a fluorescence microscope coupled with a high resolution charged coupled device camera (CCD), 40X objective or higher, GFP filter and acquisition software.
   3. Adjust the exposure time and illumination intensity to prevent oversaturated images.
   4. Capture and save images of muscles (400X magnification) from a section of the animal containing at least one complete visible oblique muscle cell. Avoid regions with a single muscle cell that is incomplete or sections that are out of focus. Also exclude images from extreme anterior and posterior regions, and regions adjacent to the vulva.
      NOTE: If the animal does not have a single visible complete muscle cell, gently slide the coverslip to turn the animal so as to expose a complete cell.
2. Measurement of muscle cell area
   1. Open the image in Fiji software (version 2.0.0 was used in this study).
   2. Use the polygon selection to carefully trace around a single oblique muscle cell. Adjust the line of the polygon at the end by dragging the anchor dot to improve tracing.
   3. Go to Analyze tab at the top of the software and click Measure to calculate the area of the selection. A separate results table containing the area and other measurements will be shown. If the area measurement is missing from the results table, go to Set Measurements under the Analyze tab and check that the area box is ticked. Likewise, untick other measurements that are not needed.
   4. For muscle cells with a degenerated/missing region, trace the missing region with the polygon selection tool and click Measure again. If there are multiple gaps within the cell, trace each gap separately.
   5. Calculate the ratio of gap area to the entire single muscle cell. A high ratio indicates a higher extent of muscle degeneration due to large gaps within the cell. If there are no missing regions, as commonly seen in wild-type animals, the ratio would be calculated as zero.
      NOTE: As a control, score also for muscle structure defects visually and compare results with the quantitative measurement. This is especially useful if the strain is being studied for the first time in the lab. For phenotypic scoring, assess the animals as defective or not-defective based on the integrity of the filaments. For example, animals with loss of distinct filament striations, GFP clumping, or gaps within muscle cells due to structural breakdown, would be scored as defective.