FE-SEM visualization of cortical microtubules in plant cells using freeze-fracture techniques

Cortical microtubules (CMTs), one of the components of cytoskeleton, control the orientation and localization of newly deposited cellulose microfibrils in cell walls, and thereby determine the shape, size, and structure of plant cells. Imaging of CMTs in plant tissues is generally performed using fluorescently labeled specimens under an optical fluorescence or confocal laser scanning microscope. However, visualizing individual CMTs by optical microscopy is challenging because the observable range is limited to superficial tissue layers accessible to light penetration. In contrast, transmission electron microscopy offers high-resolution visualization of CMTs in plant cells but is restricted to slightly oblique ultrathin sections with an approximate thickness of 70-100 nm. Given that field emission scanning electron microscopy (FE-SEM) provides both high spatial resolution and a broad field of view, the establishment of FE-SEM-based techniques for CMTs visualization is highly desirable.

Herein, we introduce a technique for visualizing CMTs within untreated plant tissues by combining two cryo-fracture techniques, freeze-knife fracture and freeze-tensile fracture, with FE-SEM. We successfully observed the arrangement of CMTs in several plant specimens, including young branches of ginkgo (Ginkgo biloba), calli from the leaves of hybrid poplar (Populus sieboldii × P. grandidentata), and root tips of the adzuki bean (Vigna angularis). CMTs were visualized on the protoplasmic fracture face (PF) using both cryo-FE-SEM and conventional room-temperature FE-SEM. While cryo-FE-SEM was employed to maintain native structures, room-temperature FE-SEM proved to be a viable alternative when specialized cryo-equipment was unavailable.

The combination of freeze-fracture techniques with FE-SEM enables the visualization of CMT arrangement in plant tissues at a high resolution and across a broad area without the need for staining or extraction of cellular components. This technique is applicable to various plant tissues and allows for detailed observation of CMTs within these tissues, providing valuable insights into the role of microtubules in the division and differentiation of plant cells.