A zWEDGI Technique to Visualize Fungal Pathogen-Infected Zebrafish Larvae

Begin with transgenic zebrafish larvae expressing fluorescent-labeled immune cells tagged with unique fluorescent markers for precise tracking.

Larvae are pre-injected with red fluorescent protein expressing Aspergillus spores, in their hindbrain.

Within the hindbrain, the fungal spores mimic local inflammation and attract immune cells, including macrophages, which phagocytose spores for their clearance.

While a few spores persist and germinate into hyphae, releasing pathogen-derived molecules and attracting neutrophils to the infection site.

Transfer an anesthetized larva into the medium-containing loading chamber of the zebrafish wounding and entrapment device for growth and imaging, or zWEDGI, device interconnected to the wounding chamber via a restriction channel.

Secure the larva within the restriction channel in a dorsal-lateral orientation for hindbrain imaging.

In the early stages, red fluorescence represents the fungal spores, while green fluorescence confirms macrophage recruitment to the infection site.

Over time, the spreading of red fluorescence signifies infection progression, while the emergence of additional blue fluorescence indicates neutrophil recruitment.