Fig. 5
Structural composition of extracellular matrix (ECM) by epifluorescence microscopy (EPM). The EPM images on 12-wells polystyrene plates incubated with RPMI and an inoculum concentration of 1 × 106 microconidia/mL at 24 h at 37 °C in vitro biofilm of Aspergillus fumigatus clinical isolate. a Co-localization of chitin/DNA. Hyphal anastomosis marked with Calcofluor white (chitin), FUN1 (fungal metabolic activity), and DAPI (DNA) (10X); b Detection of metabolic activity and chitin biofilm. Hyphal anastomosis indicated conidia marked with FUN1 and Calcofluor white. The latter exhibited a strong signal of chitin (100X); c Top view of the ECM detecting the co-localization of different molecules. Arrangement of z-stack images showing the dimensions of a section of the fungal biofilm, marked with FUN1, Calcofluor White and Flamingo stain; d Three-dimensional reconstruction of the in vitro biofilm showing molecular components of the ECM. These depicted different images that dissect the ECM and show some of its components marked as chitin (White Calcofluor, D1), hyphae with high metabolic activity (FUN1, D2) and protein (Flamingo, D3). In d, the merged image shows the reconstruction of the entire ECM model of the biofilm of A. fumigatus. All of the fluorochromes were co-localized in the ECM with the hyphae embedded within it. The ECM showed a thickness of <10 μm d. White pointed arrow: hyphae; white arrow: DNA (Signal with DAPI); white dotted circles: colocalization of exopolymers in ECM; c: conidia