A versatile microfluidic device for highly inclined thin illumination microscopy in the moss Physcomitrella patens.

High-resolution microscopy is a valuable tool for studying cellular processes, such as signalling, membrane trafficking, or cytoskeleton remodelling. Several techniques of inclined illumination microscopy allow imaging at a near single molecular level; however, the application of these methods to plant cells is limited, owing to thick cell walls as well as the necessity to excise a part of the tissue for sample preparation. In this study, we utilised a simple, easy-to-use microfluidic device for highly inclined and laminated optical sheet (HILO) microscopy using a model plant Physcomitrella patens. We demonstrated that the shallow microfluidic device can be used for long-term culture of living cells and enables high-resolution HILO imaging of microtubules without perturbing their dynamics. In addition, our microdevice allows the supply and robust washout of compounds during HILO microscopy imaging, for example, to perform a microtubule regrowth assay. Furthermore, we tested long-term (48 h) HILO imaging using a microdevice and visualised the developmental changes in the microtubule dynamics during tissue regeneration. These novel applications of the microfluidic device provide a valuable resource for studying molecular dynamics in living plant cells.