4D Widefield Fluorescence Imaging of Appressorium Morphogenesis by Magnaporthe oryzae

Fluorescence microscopy has become a widely used and indispensable tool for the M. oryzae research community, providing unique insight into appressorium formation and function. A common practice within the field is to acquire and present images of a number of different conidia, expressing a fluorescent fusion protein of interest, at various stages of infectious development, therein providing a representative "snapshot" of the population at a given point in time. Furthermore, these images typically show only a single focal plane through the specimen (2D) and therefore lack, often valuable, volumetric information. While this approach has its advantages, the continuous imaging of (multiple) single conidia in three dimensions (3D), and over time (4D), can provide additional insight into the spatial and temporal dynamics of fluorescent fusion proteins, and the subcellular structures and compartments they label, in living cells. Here we describe our typical workflow for the 4D live-cell imaging of appressorium morphogenesis in vitro using two-color widefield fluorescence microscopy and briefly outline some important considerations for strain construction, and downstream image processing and visualization.