Microscopic analysis of filamentous fungi and their interaction with immune cells

Due to their tiny habitus, their complex organisation, and their strong auto-fluorescence, fungal hyphae are not easily analysed by microscopy. Super-resolution techniques that are circumventing the resolution limit caused by the diffraction of light are of special interest for the mycological research. We evaluated the potential of diverse novel techniques in the investigation of fungal cells and their interaction with immune cells. We used direct stochastic optical reconstruction microscopy (dSTORM) to investigate the interplay of natural killer (NK) cells with Aspergillus fumigatus and the distribution of the pattern recognition receptor dectin-1 in macrophages. Furthermore, we exploited the inherent blinking behaviour of eYFP for single-molecule localisation and reconstruction to reveal the spatial distribution of the rhodopsin CarO in hyphae of Fusarium fujikuroi. In vivo confocal laser scanning microscopic (CLSM) analysis of membrane-stained murine macrophages (MH-S) revealed the time course of engulfment of fungal conidia, yielding data that are suitable for subsequent bioinformatical analysis. In order to test the influence of mutations or different treatments in the dynamics of fungal spore germination we recently developed the ImageJ toolbox HyphaTracker. This toolbox is used to process microscopic acquisitions (movies) of conidial germination. Regions of interest (ROIs) are extracted, which are analysed for their area, circularity, and timing. ROIs originating from germlings crossing other hyphae or the image boundaries are omitted during analysis. Each conidium/hypha is identified and related to its origin, thus allowing subsequent categorization. The efficiency of HyphaTracker was proofed and the accuracy was tested on simulated germling and bright field microscopic images of conidial germination of rhodopsin-deficient F. fujikuroi mutants, revealing earlier and faster germination of the CarO deficient mutant. This toolbox will also be useful in the research on pathogenic fungi to investigate the influence of drugs or mutations on the early hyphal development.