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

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.