Single-molecule detection and tracking in plants

Combining optical properties with a limited choice of fluorophores turns single-molecule imaging in plants into a challenging task. This explains why the technique, despite its success in the field of animal cell biology, is far from being routinely applied in plant cell research. The same challenges, however, also apply to the application of single-molecule microscopy to any intact tissue or multicellular 3D cell culture. As recent and upcoming progress in fluorescence microscopy will permit single-molecule detection in the context of multicellular systems, plant tissue imaging will experience a huge benefit from this progress. In this review, we address every step of a single-molecule experiment, highlight the critical aspects of each and elaborate on optimizations and developments required for improvements. We relate each step to recent achievements, which have so far been conducted exclusively on the root epidermis of Arabidopsis thaliana seedlings with inclined illumination and show examples of single-molecule measurements using different cells or illumination schemes.