Probing Aspergillus niger spore dynamics: A droplet – based microfluidic approach for single-spore analysis and stressor evaluation

Kurzfassung

Aspergillus niger spores demonstrate complex and heterogeneous responses to environmental stressors, which complicates efforts to control fungal growth and resistance. In this study, we used a droplet – based microfluidic system to encapsulate individual A. niger spores in hydrogel beads, creating a 3D microenvironment conducive to controlled, high-resolution monitoring. This platform facilitates single-cell time-lapse microscopy, enabling detailed observation of germination patterns and branching behaviors across up to 1500 spores in a single experiment. To evaluate the effects of copper ions (Cu²⁺, delivered as CuSO₄ solution) and the antifungal agent caspofungin, we measured parameters such as swelling events, swelling size, hyphal area and main hyphae length. Initial findings revealed a pronounced heterogeneity in germination and outgrowth behavior under copper stress, even in a species known for its robust copper homeostasis mechanisms. The treatment with caspofungin in comparison to led to a 175% increase in swelling size and elevated the fraction of swollen spores in wild-type strains, while exposure to a 10 mM CuSO₄ solution resulted in a reduction across all growth metrics. ICP-MS measurements of copper content before and after incubatio over 24h on chip further indicated potential uptake of Cu²⁺ ions by spores within the hydrogel matrix. Viability and cell wall integrity assessments via LIVE/DEAD fluorescence and calcofluor white staining confirmed the spores' responses to these treatments. This research highlights the nuanced, stress-induced heterogeneity in A. niger"s physiological responses and underscores the potential of microfluidic systems as powerful tools for real-time, high-throughput fungal analysis. This approach offers new insights for antifungal development and fungal behavior under metal-induced stress.