Visualizing three-dimensional fungal growth using light sheet fluorescence microscopy

The evaluation of morphology is fundamental to comprehend how fungi grow, develop, and interact with the environment. Although fungal growth has been extensively studied associated to two-dimensional geometries, lack of appropriate experimental tools has limited exploration of the complex three-dimensional (3D) structures exhibited by mycelia in more general contexts. In this paper, we report the construction of a light-sheet fluorescence microscope (LSFM) capable of performing time-lapse visualization of 3D biological structures (4D microscopy), and the use of this instrument to follow the dynamics of fungal growth. LSFM uses scanning of selective plane illumination and digital reconstruction to provide 3D images of the specimen. We describe the optical, electronic, and computational means to implement two-color LSFM, and provide detailed procedures for aligning and testing the setup. We successfully demonstrate use of both autofluorescence and specific tagging to image Trichoderma atroviride and Neurospora crassa strains growing in liquid media, over extended times (-12 h) and volumes (-400 ? 1500 ? 800 ?m3) at single-hypha resolution. The excellent image contrast provided by LSFM enables us to visualize the dynamics of mycelial architecture, interactions among hyphae, and measure rates of 3D apical extension. Altogether, our work shows a powerful imaging tool to perform 3D morphological analysis of fungi, from hyphae to mycelium.