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

Abstract

High-resolution microscopy is a valuable tool to study cellular processes, such as signalling, membrane trafficking, or cytoskeleton remodelling. Several techniques of inclined illumination microscopy allow imaging at near single molecular level; however, the application of these methods to plant cells is limited, due to thick cell walls and necessity to excise a part of the tissue for sample preparation. In this study, we developed simple, easy-to-use microfluidic device for highly inclined and laminated optical sheet (HILO) microscopy using a model plant Physcomitrella patens. We demonstrated that microfluidic device can be used to culture living cells and enables high-resolution HILO imaging of microtubules without perturbing their dynamics. In addition, our microdevice enables the supply and robust washout of compounds during HILO microscopy imaging, for example to perform 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. The microfluidic device designed in this study provides a novel tool to conduct long-term HILO microscopy and washout assays using plant cells.