An Image Processing Algorithm for Facile and Reproducible Quantification of Vomocytosis

Vomocytosis is a process that occurs when internalized fungal pathogens escape from phagocytes without compromising the viability of the pathogen and the host cell. Manual quantification of time-lapse microscopy videos is currently used as the standard to study pathogen behavior and vomocytosis incidence. However, human-driven quantification of vomocytosis (and the closely related phenomenon, exocytosis) is incredibly burdensome, especially when a large volume of cells and interactions needs to be analyzed. In this study, we designed a MATLAB algorithm that measures the extent of colocalization between the phagocyte and fungal cell (Cryptococcus neoformans; CN) and rapidly reports the occurrence of vomocytosis in a high throughput manner. Our code processes multichannel, time-lapse microscopy videos of cocultured CN and immune cells that have each been fluorescently stained with unique dyes and provides quantitative readouts of the spatiotemporally dynamic process that is vomocytosis. This study also explored metrics, such as the rate of change of pathogen colocalization with the host cell, that could potentially be used to predict vomocytosis occurrence based on the quantitative data collected. Ultimately, the algorithm quantifies vomocytosis events and reduces the time for video analysis from over 1 h to just 10 min, a reduction in labor of 83%, while simultaneously minimizing human error. This tool significantly minimizes the vomocytosis analysis pipeline, accelerates our ability to elucidate unstudied aspects of this phenomenon, and expedites our ability to characterize CN strains for the study of their epidemiology and virulence.