Imaging the delivery and behavior of cellulose synthases in Arabidopsis thaliana using confocal microscopy

Confocal microscopy has been a key tool for characterizing the behavior of cellulose synthase (CESA) proteins as they extrude cellulose into the apoplast to help construct plant cell walls. While other microscopy techniques like electron microscopy can achieve higher resolution images of CESAs, confocal microscopy is still the most accessible way to image these proteins in living plants as they are trafficked to and from the cell surface and move through the plasma membrane. Here, we describe a method for imaging fluorescently tagged CESA proteins in seedlings of Arabidopsis thaliana using spinning disk confocal microscopy, with a focus on quantifying the speed, density, and delivery rate of CESA particles. Many of these techniques can be adapted and applied to imaging other membrane-localized proteins and other plant species. In addition to imaging techniques, we describe several options for image analysis that can be optimized for different datasets.