Live Monitoring and Analysis of Fungal Growth, Viability, and Mycelial Morphology Using the IncuCyte NeuroTrack Processing Module

Abstract

Efficient live-imaging methods are pivotal to understand fungal morphogenesis, especially as it relates to interactions with host immune cells and mechanisms of antifungal drugs. Due to the notable similarities in growth patterns of neuronal cells and mycelial networks, we sought to repurpose the NeuroTrack (NT) processing module of the IncuCyte time-lapse microscopy system as a tool to quantify mycelial growth and branching of pathogenic fungi. We showed the robustness of NT analysis to study Candida albicans and five different molds and confirmed established characteristics of mycelial growth kinetics. We also documented high intra- and interassay reproducibility of the NT module for a spectrum of spore inocula and culture periods. Using GFP-expressing Aspergillus fumigatus and Rhizopus arrhizus, the feasibility of fluorescence-based NT analysis was validated. In addition, we performed proof-of-concept experiments of NT analysis for several translational applications such as studying the morphogenesis of a filamentation-defective C. albicans mutant, the effects of different classes of antifungals (polyenes, azoles, and echinocandins), and coculture with host immune cells. High accuracy was found, even at high immune cell-to-fungus ratios or in the presence of fungal debris. For antifungal efficacy studies, addition of a cytotoxicity dye further refined IncuCyte-based analysis, facilitating real-time determination of fungistatic and fungicidal activity in a single assay. Complementing conventional MIC-based assays, NT analysis is an appealing method to study fungal morphogenesis and viability in the context of antifungal compound screening and evaluation of novel immune therapeutics. IMPORTANCE Pathogenic fungi remain a major cause of infectious complications in immunocompromised patients. Microscopic techniques are crucial for our understanding of fungal biology, host-pathogen interaction, and the pleiotropic effects of antifungal drugs on fungal cell growth and morphogenesis. Taking advantage of the morphological similarities of neuronal cell networks and mycelial growth patterns, we employed the IncuCyte time-lapse microscopy system and its NeuroTrack image analysis software package to study growth and branching of a variety of pathogenic yeasts and molds. Using optimized image processing definitions, we validated IncuCyte NeuroTrack analysis as a reliable and efficient tool for translational applications such as antifungal efficacy evaluation and coculture with host immune effector cells. Hence, the IncuCyte system and its NeuroTrack module provide an appealing platform for efficient in vitro studies of antifungal compounds and immunotherapeutic strategies in medical mycology.

Live imaging and quantitative analysis of Aspergillus fumigatus growth and morphology during inter-microbial interaction with Pseudomonas aeruginosa

“…Despite the explosive growth of PA in RPMI medium resulting in considerable turbidity of the wells at high PA/AF ratios, no changes to the processing algorithms for fungal mono-culture or immune cell challenge [11] were needed to maintain high accuracy of NT analysis during co-culture with viable PA cells. Furthermore, no interference of bacterial auto-fluorescence with the image analysis algorithm was encountered.…”

“…Well plates were incubated and imaged for 18 h at 37 ° C in the IncuCyte ZOOM HD/2CLR time lapse microscopy system (Sartorius) equipped with an IncuCyte ZOOM 10 x PLAN FLUOR objective (Sartorius) as previously described [11]. Phase images were acquired hourly for all experiments.…”

“…For live cell co-culture using AF293-GFP, green fluorescence images were obtained hourly with an acquisition time of 400 milliseconds. NT image processing algorithms were applied using our published parameters for A. fumigatus and AF293-GFP [11].…”

“…Inspired by morphological and functional similarities of neuronal networks and fungal mycelia [10], we have recently proposed the IncuCyte time-lapse microscopy platform and its NeuroTrack TM (NT) image processing module as an efficient and reliable tool to study the expansion kinetics and viability of mycelial trees [11]. In this study, we hypothesized that NT analysis could present an appealing platform to quantitatively analyze mycelial morphology and expansion in the context of intermicrobial interaction.…”

Tornadic shear stress induces a transient, calcineurin-dependent hyper-virulent phenotype in Mucorales molds

“…Therefore, we conducted an array of phenotypic assays to further understand the features of TSC-induced hyper-virulence. Employing the recently adapted IncuCyte NeuroTrack time-lapse imaging approach (28), we tested whether TSC results in accelerated growth or hyphal filamentation of Mucorales. However, mycelial morphology ( Fig.…”