Automated, continuous video microscopy tracking of hyphal growth

Abstract

The growth of filamentous fungi is a complex process that involves hyphal elongation and branching. Microscopic observations provide a wealth of information on fungal growth, although often requiring laborious manual intervention to record and analyze images. Here, we introduce a novel tool for automated tracking of growth in fungal hyphae that affords quantitative analysis of growth rate and morphology. We supplied a student-grade bright field microscope with stepper motors to enable computer-control of the microscope stage. In addition, we developed an image-processing routine that detects in real-time the tip of a hypha and tracks it as the hypha elongates. To achieve continuous observation of hyphal growth, our system automatically maintains the observed sample within field-of-view and performs periodic autofocus correction in the microscope. We demonstrate automated, continuous tracking of hyphal growth in Trichoderma atroviride with sampling rates of seconds and observation times of up to 14 h. Tracking records allowed us to determine that T. atroviride hyphae grow with characteristic elongation rates of ∼70 nm/s. Surprisingly, we found that prior to the occurrence of an apical branching event the parental hypha stopped growing during a few minutes. These arrest events presented occasionally for subapical branching as well. Finally, from tracking data we found that the persistence length (a measure of filament extension before presenting a change in direction) associated to T. atroviride hyphae is 362 µm. Altogether, these results show how integration of image analysis and computer control enable quantitative microscopic observations of fungal hyphae dynamics.