Microscopy of fungal biofilms

•Fixed biofilms may be clarified for deep imaging by refractive index matching.•For the biofilms discussed in this review, optimal clarity is obtained for n>1.52.•In this range, oil-immersion optics minimize depth-dependent spherical aberration.•Optical sectioning microscopy with long focusing range is almost always required.•For clearing, both an embedding protocol and an immersion protocol are detailed. Fungal biofilms are heterogeneous, surface-associated colonies comprised of filamentous hyphae (chains of elongated cells), pseudohyphal cells, yeast-form cells, and various forms of extracellular matrix. When grown on a substratum under liquid culture medium, the microbial fungus Candida albicans forms dense biofilms that range in thickness from 100 to 600μm. Apical hyphae in the medium and invasive hyphae in the substratum may add greatly to the thickness and complexity of the biofilm. Because of the heterogeneity of the structure, and the large refractive index differences between cell walls, cytoplasm, and medium, fungal biofilms appear optically opaque. For fixed specimens that can be transferred out of an aqueous medium, refractive index matching methods provide a high degree of clarification. Confocal scanning, 2-photon scanning, or selective-plane illumination microscopy then can be used to obtain high-quality image data spanning the full thickness of the biofilm. Using refractive index matching and confocal microscopy, we have imaged many interesting features within wild-type, mutant, and engineered biofilms, including cellular phenotypes that vary with position, the effect of growth conditions, and gene expression through reporter constructs. This approach greatly expands the range of microscopical studies, allowing researchers to observe and quantify specific phenomena within medically or industrially relevant forms of microbial growth.