Enhancing the blinking fluorescence of single-molecule localization imaging by using a surface-plasmon-polariton-enhanced substrate

Super-resolution imaging based on single-molecule localization microscopy combined with the surface plasmon polariton (SPP)-enhanced fluorescence of spontaneously blinking fluorophores was demonstrated to visualize the nanoscale-level positioning information of cell-adhesion-associated proteins. Glass substrates with a deposited silver layer were utilized to induce a SPP-enhanced field on the silver surface and significantly strengthen the fluorescence signals of the fluorophores by more than 300%. The illumination power density for localization imaging at a spatial resolution of 25 ± 11 nm was 31.6 W cm-2. This low illumination power density will facilitate the reduction of phototoxicity of the biospecimens for single-molecule localization imaging. The proposed strategy provides a uniform distribution of the SPP-enhanced field on the silver surface, enabling visualization of the spatial distribution of labeled proteins without interference caused by the enhanced field distribution.