Modular Design of Membrane-Impermeable Versatile Probe for Specific Imaging of Cell Walls and Real-Time Detection of Cell Membrane Damage

The versatile fluorescent dyes are essential for specifically labeling plant cell walls in vivo, monitoring plasma membrane damage, and assessing cell viability. However, such dyes are rare and often discovered accidentally due to a lack of design principles. Propidium iodide, a well-known example, has limitations like low brightness, high toxicity, and poor bacterial differentiation. To address these challenges, we developed VersaDye, a modular probe designed for specific imaging of live plant cell walls and monitoring plasma membrane damage in plant cells, human cells, and certain bacteria. The design integrates impermeability principles and environment-dependent fluorophore scaffolds. VersaDye enables bright, wash-free labeling of plant cell walls and can stain various plant organs for constructing 3D tissue organization. Notably, it can selectively distinguish live Gram-positive from Gram-negative bacteria, a feature absent in other dyes. Its impermeability and targeting ability also allow it to probe membrane damage caused by physical, chemical, and biological stimuli. This study marks the first use of VersaDye in analyzing cell damage in live plants under salt stress. VersaDye offers a robust platform for wash-free, in vivo membrane damage monitoring and simultaneous cell wall labeling. Additionally, its design suggests adaptability for regulating permeability to meet specific diagnostic needs, such as identifying membrane-compromised cells in diseases or enabling high-throughput antibiotic screening targeting specific bacteria.