Step 1. Mount Worms on Agar Pad (Timing ~2 min)
Mount transgenic worms (laminin::Dendra or type IV collagen::Dendra) of appropriate developmental stage by picking worms into a 1.5µl drop of M9 on a 5% noble agar pad [with 0.01 M of the anesthetic sodium azide (NaN3)] atop a standard microscope slide. Cover with standard glass coverslip and proceed to step 2.
Helpful Suggestion: If examining a specific developmental stage, it may be beneficial to perform synchronized L1 stage larval plating, which facilitates picking the appropriate developmental stage.
Step 2. Photoconversion using a 405nm Ultra Violet (UV) Laser (Timing ~5-10 min)
Using a laser scanning confocal microscope equipped with a 63X or 100X objective, acquire an image of the worm you wish to photoconvert. Next, photoconvert a region(s) of BM using a customizable region of interest (ROI) bleaching tool within your laser scanning confocal acquisition software. For our studies of the uterine-vulval BM in the late L3 stage of hemaphrodite development, the optimal photoconversion was achieved using the 405nm laser at 1-2% power, and a high number (100-200) of iterations (See Figure 1 for laminin::Dendra and Figure 2 for type IV collagen::Dendra). Depending on your experimental design, you should be able to photconvert several regions within the same animal or a few worms per slide. When photoconverting several worms per slide it may be useful to carefully mark particular worms so that you can easily keep track of them when moving between microscopes. If working on an inverted microscope, this can be done with a marker on the underneath side of the microscope slide (opposite the coverslip).
Troubleshooting: You may need to adjust the laser power or the number of iterations to optimize photoconversion of the BM in your context.
Troubleshooting: Using the laser scanning confocal, if there is BM above and below the focal plane, some Dendra2 will become photoconverted and may give undesirable results or interfere with experimental analysis. Reducing the pinhole size on the confocal can minimize this problem.
Troubleshooting: Complete this and the following step as quickly as possible in order for the worms to remain healthy. If the worms are on the NaN3 pad for longer than 15-20 min., they will likely not survive. After performing the procedure a few times, you will become familiar with the protocol and more efficient with your time.
Step 3. Image Worms Immediately following Photoconversion (Timing ~2 min)
If appropriate for your experiment, image worms on a wide field fluorescence microscope or spinning disc confocal. Be sure to use the same image acquisition settings (i.e, laser power, exposure, gain, binning, etc.), if you plan to do quantitative measurements amongst different images.
Troubleshooting: Though sufficient, the signal from the photoconverted BM may be relatively weak (more so with type IV collagen::Dendra). If your experimental design requires you to image the worm at multiple time points or to acquire z-stacks, it would be best to use a spinning disc confocal, which uses less light than the laser scanning confocal.
Step 4. Recover Worms to Food Plates (Timing ~1 min)
Recover worms by sliding coverslip back and picking (with a glob of OP50) or using a mouth pipette and M9 to transfer animals to food (OP50 E. coli) plates for the necessary amount a time at the appropriate temperature (e.g., 15˚, 20˚, or 25˚C). For example, your experimental design might require L3 larval stage worms to develop for 4 hours at 25˚C before you reexamine them.
Step 5. Mount Worms and Reimage (Timing ~5-10 min)
If appropriate, mount worms on new noble agar pad with 0.01M NaN3 and reimage. Image analysis softwares (e.g., NIH ImageJ, Imaris by Bitplane, or Volocity by Improvision) can be used to quantify changes in the position or fluorescence intensities of regions of photoconverted BM in 3D, over time. As a reminder, if taking quantitative measurements, it will be important to ensure that image acquisition setting (e.g., exposure, gain, laser power, binning, etc.) are maintained between imaging sessions or experiments.