Visualization of Aspergillus fumigatus biofilms with Scanning Electron Microscopy and Variable Pressure-Scanning Electron Microscopy: A comparison of processing techniques

- Visualization of A. fumigatus biofilms is influenced by SEM processing techniques.
- Hyphae and ECM are sensitive to interface, reagents, fixation times and drying.
- Shortened primary fixation, with OsO4 staining and CPD, optimally preserves structure.
- Contrasting with OsO4, RuO4 and Ruthenium Red enhance resolution for VP-SEM.
- Ionic Liquid treatment renders biofilms hydrated and conductive for SEM analysis.

Aspergillus fumigatus biofilms consist of a three-dimensional network of cellular hyphae and extracellular matrix. They are involved in infections of immune-compromised individuals, particularly those with cystic fibrosis. These structures are associated with persistence of infection, resistance to host immunity, and antimicrobial resistance. Thorough understanding of structure and function is imperative in the design of therapeutic drugs. Optimization of processing parameters, including aldehyde fixation, heavy metal contrasting, drying techniques and Ionic Liquid treatment, was undertaken for an ultrastructural approach to understand cellular and extracellular biofilm components. Conventional and Variable Pressure Scanning Electron Microscopy were applied to analyze the structure of biofilms attached to plastic and formed at an air-liquid interface.