Live Imaging of Macrophage Cell Death During Mycobacterial Infection in Zebrafish Embryos

Begin with transgenic zebrafish embryos expressing fluorescently labeled macrophages, mounted in low-melting agarose in an imaging dish.

These embryos are infected with fluorescently labeled Mycobacterium marinum by intramuscular microinjection into the midbrain, initiating a localized immune response.

Cover the solidified agarose with embryo media containing anesthetic drugs to maintain anesthesia and chemical inhibitors to suppress pigmentation, ensuring optical clarity for imaging.

Place the dish in the environmental chamber of a high-resolution confocal microscope.

Using bright-field illumination, locate the infected region.

Activate the fluorescence laser channels, set the Z-stack start and end positions, and initiate time-lapse imaging.

Visualize the infected macrophage, filled with fluorescent bacteria, as it becomes rounded and immobile.

This indicates cytoskeletal breakdown and the onset of lytic cell death.

Over time, the macrophage swells and ruptures, releasing cytoplasmic contents and internalized bacteria into the surrounding tissue, a hallmark of inflammatory lytic cell death.

Once the agarose has completely solidified, cover the agarose with a layer of egg water. After setting up the environmental chamber, place the 35 millimeter glass-bottom dish with the zebrafish in the environmental chamber. Open the 405 diode, argon at 20% power, and DPSS 561 nanometer laser. Set up the appropriate laser power in spectrum settings.

Choose the "XYZ" "Sequential Scan" acquisition mode, and set images format to "512 by 512 pixels". Switch to "Live Data Mode", target the position of the first zebrafish, and mark the "Begin" and "End" Z position. Repeat this process for each of the remaining embryos. Add a "Pause" at the end of the program. Define the loop and cycle of the program, and save the file.