Ultra-photostable fluorescent dye molecular engineering—for measuring plant cells’ membrane-spacing through a “deposition-embedding” strategy

Abstract

The plant cell membrane serves as a barrier, isolating the cell's interior from its external environment. Unlike animal cells, where the cytoplasmic membrane can be easily fluorescently labeled through genetic engineering, plant cells often rely more heavily on small molecule fluorescent probes to address the problem of probe internalization. Meanwhile, due to cellular internalization, current plasma fluorescent probes struggle to stain cell membranes for long periods of time. In addition, these probes tend to accumulate in the cell wall, making it impossible to achieve specific, high-noise-to-noise staining of cell membranes. In response to these challenges, we propose a novel “deposition-embedding” strategy for developing a plant cell membrane probe. The compound PTBT-O-NPh₂, with its low solubility and high hydrophobicity, is designed to limit membrane penetration. Instead, it rapidly deposits on the membrane surface and embeds itself into the lipid environment via strong hydrogen bonding with phospholipid molecules. Additionally, its exceptional resistance to photobleaching and long-term retention capability allow it to measure membrane-spacing over a period of 120 hours. These findings suggest that the “deposition-embedding” strategy could be instrumental in developing a new generation of fluorescent dyes for studying plant mechanobiology.