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

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.