Live microscopy analysis of endosomes and vesicles in tip-growing root hairs

Tip growth is one of the most preferable models in the study of plant cell polarity; cell wall deposition is restricted mainly to a certain area of the cell, and cell expansion at this specific area leads to the development of tubular outgrowth. Tip-growing root hairs are well-established systems for such studies, because their lateral position within the root makes them easily accessible for experimental approaches and microscopic observations. Fundamental structural and molecular processes driving tip growth are exocytosis, endocytosis, and all aspects of vesicular and endosomal dynamic trafficking, as related to targeted membrane flow. Study of vesicles and endosomes in living root hairs, however, is rather difficult, due to their small size and due to the resolution limits of conventional light microscopes. Here we present noninvasive approaches for visualizing vesicular and endosomal compartments in the tip of growing root hairs using electronic light microscopy, contrast-enhanced video light microscopy, and confocal laser scanning microscopy (CLSM). These methods allow utilizing the maximum resolution of the light microscope. Together with protocols for appropriate preparation of living plant samples, the described methods should help improve our understanding on how tiny vesicles and endosomes support the process of tip growth in root hairs.