Long-term imaging of three-dimensional hyphal development using the ePetri dish

Imaging three-dimensional microbial development and behavior over extended periods is crucial for advancing microbiological studies. Here, we introduce an upgraded ePetri dish system specifically designed for extended microbial culturing and 3D imaging, addressing the limitations of existing methods. Our approach includes a sealed growth chamber to enable long-term culturing, and a multi-step reconstruction algorithm that integrates 3D deconvolution, image filtering, ridge, and skeleton detection for detailed visualization of the hyphal network. The system effectively monitored the development of Aspergillus brasiliensis hyphae over a seven-day period, demonstrating the growth medium’s stability within the chamber. The system’s 3D imaging capability was validated in a volume of 5.5 mm × 4 mm × 0.5 mm, revealing a radial growth pattern of fungal hyphae. Additionally, we show that the system can identify potential filter failures that are undetectable with 2D imaging. With these capabilities, the upgraded ePetri dish represents a significant advancement in long-term 3D microbial imaging, promising new insights into microbial development and behavior across various microbiological research areas.