Clearing techniques for deeper imaging of plants and plant–microbe interactions

Plant cells are uniquely characterized by exhibiting cell walls, pigments, and phenolic compounds, which can impede microscopic observations by absorbing and scattering light. The concept of clearing was first proposed in the late nineteenth century to address this issue, aiming to render plant specimens transparent using chloral hydrate. Clearing techniques involve chemical procedures that render biological specimens transparent, enabling deep imaging without physical sectioning. Drawing inspiration from clearing techniques for animal specimens, various protocols have been adapted for plant research. These procedures include (i) hydrophobic methods (e.g., Visikol™), (ii) hydrophilic methods (ScaleP and ClearSee), and (iii) hydrogel-based methods (PEA-CLARITY). Initially, clearing techniques for plants were mainly utilized for deep imaging of seeds and leaves of herbaceous plants such as Arabidopsis thaliana and rice. Utilizing cell wall-specific fluorescent dyes for plants and fungi, researchers have documented the post-penetration behavior of plant pathogenic fungi within hosts. State-of-the-art plant clearing techniques, coupled with microbe-specific labeling and high-throughput imaging methods, offer the potential to advance the in planta characterization of plant microbiomes.