Division of Advanced Materials, Instituto Potosino de Investigación Científica y Tecnológica, Camino a la Presa San José 2055, 78216 San Luis Potosí, Mexico.
Division of Molecular Biology, Instituto Potosino de Investigación Científica y Tecnológica, Camino a la Presa San José 2055, 78216 San Luis Potosí, Mexico.
Fungal Genet Biol. 2019 Feb;123:25-32. doi: 10.1016/j.fgb.2018.11.006. Epub 2018 Nov 30.
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.
丝状真菌的生长是一个复杂的过程，涉及菌丝的伸长和分支。显微镜观察为真菌生长提供了丰富的信息，尽管通常需要费力的手动干预来记录和分析图像。在这里，我们引入了一种用于自动跟踪丝状真菌生长的新工具，该工具可对生长速率和形态进行定量分析。我们为学生级别的明场显微镜配备了步进电机，以实现显微镜载物台的计算机控制。此外，我们开发了一种图像处理程序，该程序可以实时检测菌丝的尖端，并在菌丝伸长时跟踪它。为了实现对菌丝生长的连续观察，我们的系统自动将观察到的样本保持在视场范围内，并在显微镜中进行周期性的自动对焦校正。我们展示了在深绿木霉中自动、连续跟踪菌丝生长的过程，采样率为秒级，观察时间长达 14 小时。跟踪记录使我们能够确定深绿木霉菌丝以特征性的伸长速率（约 70nm/s）生长。令人惊讶的是，我们发现，在发生顶端分支事件之前，亲代菌丝在几分钟内停止生长。这些停滞事件偶尔也会出现在亚顶端分支处。最后，从跟踪数据中，我们发现与深绿木霉菌丝相关的持久长度（在方向发生变化之前延伸细丝的长度）为 362µm。总之，这些结果表明，图像分析和计算机控制的集成如何使真菌菌丝动态的定量显微镜观察成为可能。
