Abstract
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.
Highlights
Observing protein dynamics in living cells at a single-molecule level provides researchers with valuable data on protein interactions and their function
total internal reflection fluorescent (TIRF) can be applied only when the molecules of interest are very close to the cell surface, which inevitably limits the TIRF use in plant cells surrounded by thick cell walls
We test and optimise the compatibility of a microfluidic device for highly inclined and laminated optical sheet (HILO) imaging in the moss Physcomitrella patens, focusing on microtubule dynamics
Summary
Observing protein dynamics in living cells at a single-molecule level provides researchers with valuable data on protein interactions and their function. TIRF can be applied only when the molecules of interest are very close to the cell surface, which inevitably limits the TIRF use in plant cells surrounded by thick cell walls. Another similar approach is variable angle evanescent microscopy (VAEM), known as ‘pseudo-TIRF’, oblique illumination fluorescent microscopy or highly inclined and laminated optical sheet (HILO) microscopy, which allows to image events more distant from the cell boundary[4]. TIRF and HILO share certain similarities and the major difference between them is the angle of excitation beam[5] For both imaging methods, the sample surface has to be flattened against the coverslip. We were able to perform a long-term (48 h) HILO imaging using a microdevice to visualise the developmental changes in the cortical microtubules array during tissue regeneration
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