Super-resolution imaging of microtubules in Medicago sativa

Abstract Study of microtubules on cellular and subcellular levels is compromised by limited resolution of conventional fluorescence microscopy. However, it is possible to improve Abbe's diffraction-limited resolution by employment of super-resolution microscopy methods. Two of them, described herein, are structured-illumination microscopy (SIM) and Airyscan laser scanning microscopy (AM). Both methods allow high-resolution imaging of cortical microtubules in plant cells, thus contributing to the current knowledge on plant morphogenesis, growth and development. Both SIM and AM provide certain advantages and characteristic features, which are described here. We present immunofluorescence localization methods for microtubules in fixed plant cells achieving high signal efficiency, superb sample stability and sub-diffraction resolution. These protocols were developed for whole-mount immunolabeling of root samples of legume crop species Medicago sativa. They also contain tips for optimal sample preparation of plants germinated from seeds as well as plantlets regenerated from somatic embryos in vitro. We describe in detail all steps of optimized protocols for sample preparation, microtubule immunolabeling and super-resolution imaging.