Microbial dysbiosis in the gut and immune system dysfunctions are often associated with the pathogenesis of opportunistic infections, chronic inflammation, and inflammatory bowel disease [1,2]. Traditional in vitro models proposed to study the underlying mechanisms lack the required tissue complexity and therefore often fail to accurately reproduce the complex, multi-component structure of the intestinal wall. On the other hand, animal models are not always transferable to the human condition, as there are significant differences in the immune system and microbiota between humans and animals such as mice [3].
To effectively investigate human-microbial interactions in a gut-like environment, a three-dimensional micro-physiological model was established to mimic conditions within the intestine. This gut-on-chip model includes a bloodstream-like compartment with endothelial cells, an intestinal epithelial barrier including resident innate immune cells and a perfused three-dimensional tissue structure of villi and crypts [4].
We apply an image-based approach to analyze colonization and infection with the opportunistic pathogenic fungus Candida albicans in the gut-on-chip model. In this model, the fungus switches to hyphal growth, eventually forming microcolonies. After 12 h of infection, cells are fixed. C. albicans are stained with an anti-Candida antibody and the nuclei of epithelial cells are stained with DAPI to visualize tissue architecture. Three-dimensional multi-channel images are acquired utilising laser scanning confocal microscopy. We developed an automated image analysis pipeline, which provides three-dimensional geometrical and morphological characterization of microcolonies, including the surface-to-volume ratio, sphericity and density. Furthermore, the complexity of microcolony surfaces is quantified using the concept of fractal dimension. Three-dimensional multi-colocalization analysis is performed to quantify the interaction between microcolonies and different tissue structures, as well as the level of fungal invasion into the endothelial compartment.
The combination of the gut-on-chip model and this image-based approach facilitate quantitative study of C. albicans infection patterns and dissection of the role of fungal and host factors during infection in situ in a complex in vivo-like environment.

References
[1] I.D., Iliev; and I. Leonardi; Nature Reviews Immunology, Fungal dysbiosis: immunity and interactions at mucosal barriers, 2017, 17(10), pp.635-646.
[2] X.C., Morgan; T.L., Tickle; H., Sokol; D., Gevers; K.L., Devaney; D.V., Ward; J.A., Reyes; S.A., Shah; N., LeLeiko; , S.B., Snapper and A., Bousvaros, Genome biology, Dysfunction of the intestinal microbiome in inflammatory bowel disease and treatment, 2012, 13(9), pp.1-18.
[3] T.L.A., Nguyen; S., Vieira-Silva; A., Liston and J. Raes, Disease models & mechanisms, How informative is the mouse for human gut microbiota research?, 2015, 8(1), pp.1-16.
[4] M., Maurer; M.S,. Gresnigt; A., Last; T., Wollny; F., Berlinghof; R., Pospich; Z., Cseresnyes; A., Medyukhina; K., Graf; M., Groeger; M., Raasch; F, Siwczak; S, Nietzsche; I.D. Jacobsen; M.T. Figge; B. Hube; O. Huber; and A.S. Mosig, Biomaterials, A three-dimensional immunocompetent intestine-on-chip model as in vitro platform for functional and microbial interaction studies, 2019, 220, p.119396.