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

Abstract

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.

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

“…The refractive indexes of the biolayer assembled using thiol (or silane) and fibronectin, plasma membrane, and cytoplasm were set as 1.46, 1.46, and 1.36, respectively. 25,42 The biolayer and plasma membrane were both assumed to have thicknesses of 10 nm. 25,43 Figure 1b illustrates the reflectivity of the glass coverslip and silverlayer substrate with silver-layer thicknesses of 30, 37, 44, and 51 nm at various incident angles.…”

“…25,42 The biolayer and plasma membrane were both assumed to have thicknesses of 10 nm. 25,43 Figure 1b illustrates the reflectivity of the glass coverslip and silverlayer substrate with silver-layer thicknesses of 30, 37, 44, and 51 nm at various incident angles. The SPP on the silver-layer substrates of different thicknesses in the cell-attached condition could be induced at similar incident angles at a range of 73.38−73.62°in the four silver layers.…”

“…The field enhancement and SPCE angle also depend on the excitation wavelength of the laser, the emission wavelength of the fluorophores, the refractive index of the surrounding medium, and the thickness of the silver layer . Moreover, the detected SPCE fluorescence intensity ( I f ) exhibited a proportional relationship with the product of the power of excitation light, local electric field enhancement, modified quantum yield ( Q m ), and emission coupling yield ( Y ec ), which can be represented as follows: where P ex is the excitation power, and E en and E i are the local electric field with and without SPP enhancement, respectively. ,,− Consequently, the simulation analysis of the local field enhancement, modified quantum yield, and emission coupling yield at various silver-layer thicknesses was adopted to determine the optimal SPCE fluorescence of each single-molecule localization event from the cell proteins labeled with spontaneously blinking fluorophores using an incident p-polarized light at a wavelength of 638 nm in SMLM. Figure a presents an SPCE substrate with a five-layer (0/1/2/3/4) structure comprising a glass coverslip, silver layer, biolayer, plasma membrane, and cytoplasm.…”

“…Consequently, an alternative approach involves adopting a flat metal thin-film substrate, creating a surface plasmon polariton (SPP) on its surface and using the uniform distribution of the SPP-enhanced field to increase the blinking fluorescence of the spontaneously blinking fluorophores. 25 Through the combination of these two strategies, focal adhesions labeled with the HMSiR dye on SPP-enhanced substrates have enabled a fluorescence enhancement of 300% and a localization precision of 25 nm at a low excitation power density of 31.6 W cm −2 in SMLM imaging. However, the configuration of two opposite objective lenses in SPPenhanced SMLM increases the difficultly of use and limits its utility.…”

Photoluminescence control by hyperbolic metamaterials and metasurfaces: a review

“…Detection schemes with fluorescence markers attached to analytes are routinely used in clinical diagnosis. Moreover, for optoelectronic emitting devices it is desirable to enhance their PL emission to improve efficiency of light sources, such as LEDs, lasers, and single-photon sources, since PL is the essential part of their emitting schemes 11,12 . This is the main motivation behind many of the recent developments in engineering spontaneous emission using nanoscale structures.…”