title: Long-Time Plasma Membrane Imaging Based on a Two-Step Synergistic Cell Surface Modification Strategy.
abstract: Long-time stable plasma membrane imaging is difficult due to the fast cellular internalization of fluorescent dyes and the quick detachment of the dyes from the membrane. In this study, we developed a two-step synergistic cell surface modification and labeling strategy to realize long-time plasma membrane imaging. Initially, a multisite plasma membrane anchoring reagent, glycol chitosan-10% PEG2000 cholesterol-10% biotin (abbreviated as "GC-Chol-Biotin"), was incubated with cells to modify the plasma membranes with biotin groups with the assistance of the membrane anchoring ability of cholesterol moieties. Fluorescein isothiocyanate (FITC)-conjugated avidin was then introduced to achieve the fluorescence-labeled plasma membranes based on the supramolecular recognition between biotin and avidin. This strategy achieved stable plasma membrane imaging for up to 8 h without substantial internalization of the dyes, and avoided the quick fluorescence loss caused by the detachment of dyes from plasma membranes. We have also demonstrated that the imaging performance of our staining strategy far surpassed that of current commercial plasma membrane imaging reagents such as DiD and CellMask. Furthermore, the photodynamic damage of plasma membranes caused by a photosensitizer, Chlorin e6 (Ce6), was tracked in real time for 5 h during continuous laser irradiation. Plasma membrane behaviors including cell shrinkage, membrane blebbing, and plasma membrane vesiculation could be dynamically recorded. Therefore, the imaging strategy developed in this work may provide a novel platform to investigate plasma membrane behaviors over a relatively long time period.
authors.affiliation: State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University , Nanjing 210096, P. R. China.
authors.forename: Hao-Ran
authors.initials: HR
authors.lastname: Jia
authors.affiliation: State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University , Nanjing 210096, P. R. China.
authors.forename: Hong-Yin
authors.initials: HY
authors.lastname: Wang
authors.affiliation: Key Laboratory of Bioorganic Phosphorous Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University , Beijing 100084, P. R. China.
authors.forename: Zhi-Wu
authors.initials: ZW
authors.lastname: Yu
authors.affiliation: Department of Chemistry, University of Michigan , 930 North University Avenue, Ann Arbor, Michigan 48109, United States.
authors.forename: Zhan
authors.initials: Z
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University , Nanjing 210096, P. R. China.
authors.forename: Fu-Gen
authors.initials: FG
authors.lastname: Wu
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*
chemical_list: D016650:Fluorescein-5-isothiocyanate