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Single-Molecule Blinking Fluorescence Enhancement by Surface Plasmon-Coupled Emission-Based Substrates for
Fan-Ching Chien1, Chun-Yu Lin2, Gerald Abrigo1
1Department of Optics and Photonics, National Central University, Taoyuan 32001, Taiwan.
Analytical Chemistry
|November 3, 2021
Summary
Fabricated surface plasmon-coupled emission (SPCE) substrates significantly enhance fluorescence for single-molecule localization microscopy (SMLM). These SPCE substrates improve fluorophore detection and reduce excitation power, enabling nanoscale imaging of proteins like EGFRs.
Area of Science:
- Nanotechnology
- Biophysics
- Microscopy
Background:
- Single-molecule localization microscopy (SMLM) requires high excitation power and is limited by fluorophore blinking.
- Visualizing protein distribution on cell membranes at the nanoscale is crucial for understanding cellular processes.
Purpose of the Study:
- To fabricate and characterize surface plasmon-coupled emission (SPCE) substrates for enhanced SMLM.
- To reduce excitation power density and improve the detection of spontaneously blinking fluorophores.
- To visualize nanoscale protein distribution on plasma membranes.
Main Methods:
- Fabrication of glass coverslip substrates coated with silver layers of varying thicknesses.
- Systematic investigation of local field enhancement, quantum yield, and emission coupling.
- SMLM imaging of fluorophore-labeled epidermal growth factor receptors (EGFRs) on A549 lung cancer cells.
Main Results:
- A 44 nm silver layer optimized SPCE, yielding a 480% fluorescence enhancement and directional emission.
- Reduced excitation power density to 23 W cm⁻² for SMLM.
- Achieved a localization precision of 19 ± 7 nm for EGFRs.
- Demonstrated reduced EGFR abundance on plasma membranes upon PIKfyve inhibitor treatment.
Conclusions:
- SPCE substrates significantly improve SMLM performance by enhancing fluorescence and reducing power requirements.
- Optimized SPCE substrates enable high-resolution visualization of protein distribution on cell membranes.
- SPCE-SMLM is a powerful tool for studying protein dynamics and drug effects at the nanoscale.
