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 μm³) 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.

形态学评估是理解真菌如何生长、发育以及与环境相互作用的基础。尽管已经对与二维几何形状相关的真菌生长进行了广泛的研究，但由于缺乏合适的实验工具，对菌丝体在更一般情况下表现出的复杂三维 (3D) 结构的探索受到了限制。在本文中，我们报告了能够对 3D 生物结构（4D 显微镜）进行延时可视化的光片荧光显微镜 (LSFM) 的构建，以及使用该仪器跟踪真菌生长的动态。LSFM 使用选择性平面照明扫描和数字重建来提供样本的 3D 图像。我们描述了实现双色 LSFM 的光学、电子和计算方法，并提供校准和测试设置的详细程序。我们成功地展示了对图像的自发荧光和特定标记的使用Trichoderma atroviride和Neurospora crassa菌株在液体培养基中以单菌丝分辨率在较长时间（~12 小时）和体积（~400 × 1500 × 800 μm ³）中生长。LSFM 提供的出色图像对比度使我们能够可视化菌丝结构的动态、菌丝之间的相互作用以及测量 3D 顶端延伸率。总而言之，我们的工作展示了一种强大的成像工具，可以对从菌丝到菌丝体的真菌进行 3D 形态分析。