Single-Molecule Blinking Fluorescence Enhancement by Surface Plasmon-Coupled Emission-Based Substrates for Single-Molecule Localization Imaging

Fan Ching Chien, Chun Yu Lin, Gerald Abrigo

研究成果: 雜誌貢獻期刊論文同行評審

4 引文 斯高帕斯(Scopus)

摘要

Surface plasmon-coupled emission (SPCE) substrates to enhance the blinking fluorescence of spontaneously blinking fluorophores in single-molecule localization microscopy (SMLM) were fabricated to reduce the excitation power density requirement and reveal the distribution of fluorophore-labeled proteins on a plasma membrane with nanoscale-level resolution. The systemic investigation of the contribution of local field enhancement, modified quantum yield, and emission coupling yield through glass coverslip substrates coated with metal layers of different thicknesses revealed that the silver-layer substrate with a thickness of 44 nm produces the highest SPCE fluorescence in spontaneously blinking fluorophores, and it has a highly directional SPCE fluorescence, which helps improve the detection efficiency. Moreover, the uniform and surface-enhanced field created on the substrate surface is beneficial for fluorescence background reduction in single fluorophore detection and localization, as well as for revealing the real position of fluorophores. Consequently, compared with a glass coverslip substrate, the presented SPCE substrate demonstrated a fluorescence enhancement of 480% and an increase in blinking events from a single spontaneously blinking fluorophore; moreover, the required excitation power density for SMLM imaging was significantly reduced to 23 W cm-2 for visualizing the distribution of epidermal growth factor receptors (EGFRs) on the basal plasma membrane of A549 lung cancer cells with a localization precision of 19 ± 7 nm. Finally, the fluorophore-labeled EGFRs on the basal plasma membrane in the presence of PIKfyve-specific inhibitor treatment were explored using SPCE-SMLM imaging; the results revealed a distinct reduction in the density of localization events because of a decrease in EGFR abundance at the plasma membranes of the cells.

原文???core.languages.en_GB???
頁(從 - 到)15401-15411
頁數11
期刊Analytical Chemistry
93
發行號46
DOIs
出版狀態已出版 - 23 11月 2021

指紋

深入研究「Single-Molecule Blinking Fluorescence Enhancement by Surface Plasmon-Coupled Emission-Based Substrates for Single-Molecule Localization Imaging」主題。共同形成了獨特的指紋。

引用此