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

Abstract

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.

Temporal‐Focusing Multiphoton Excitation Single‐Molecule Localization Microscopy Using Spontaneously Blinking Fluorophores

“…Consequently, HEtetTFER demonstrated spontaneous switching between the signals of fluorescence‐on and fluorescence‐off, as depicted by TPE fluorescence images (inset in Figure 2a). To estimate the fluorescence intensity and blinking events of HEtetTFER, the TPE fluorescence signals of HEtetTFER were detected at an intensity threshold of $${{I}_{{\rm t}{\rm h}}={I}_{{\rm a}{\rm v}{\rm g}}+3{I}_{{\rm s}{\rm t}{\rm d}}}$$ , where $${{I}_{{\rm a}{\rm v}{\rm g}}}$$ and $${{I}_{{\rm s}{\rm t}{\rm d}}}$$ are the average and standard deviation of the background intensity, respectively [44] . Signals exceeding the threshold were regarded as indicative of TPE fluorescence of HEtetTFER and were used to obtain the histograms of blinking intensity and determine the number of blinking events (Figures 2b and 2c).…”

“…To estimate the fluorescence intensity and blinking events of HEtetTFER, the TPE fluorescence signals of HEtetTFER were detected at an intensity threshold of I th ¼ I avg þ 3I std , where I avg and I std are the average and standard deviation of the background intensity, respectively. [44] Signals exceeding the threshold were regarded as indicative of TPE fluorescence of HEtetTFER and were used to obtain the histograms of blinking intensity and determine the number of blinking events (Figures 2b and 2c). The average TPE fluorescence blinking intensity and the average number of blinking events from a single HEtetTFER fluorophore were 722 a.u.…”

Metal–Dielectric Interfacial Engineering with Mesoporous Nano-Carbon Florets for 1000-Fold Fluorescence Enhancements: Smartphone-Enabled Visual Detection of Perindopril Erbumine at a Single-molecular Level

“…These studies unambiguously pointed to the advantages of the engineered morphologies with sharp edges (such as nanostars, nanoprims, and nanorods) and their hierarchical assemblies for providing high electromagnetic (EM) field confinement, leading to enhanced SPCE. − Our earlier studies in this direction contributed to this by reaffirming the structural advantages of nanovoids and nano-cavities for achieving significant plasmon-based enhancements. − These, in turn, have been utilized for ultrasensitive and reliable analytical detection through both vibrational Raman (SERS) and fluorescence (SPCE). − Furthermore, 118-fold SPCE enhancements have been demonstrated by minimizing Ohmic losses using high refraction index dielectrics in conjugation with metal nanostructures . In spite of such significant advancements in both fundamental aspects and the applications thereof, the major challenges in SPCE enhancements using metallic nanostructures have been (i) inevitable Ohmic losses along with radiative damping, ,, (ii) poor chemical stability, especially in real-time applications, ,, and (iii) extensive isotropic photon scattering that compromises the magnitude of SPCE enhancements. − As opposed to the variety of metal, non-metallic, dielectric, two-dimensional, and zero-dimensional substrates that have been investigated for SERS, substrates for SPCE have been primarily restricted to metallic and their composites. ,, Thus, a ubiquitous platform that synergistically couples the plasmonic advantages of metallic nanostructures while simultaneously minimizing the Ohmic and radiative losses is desirable for improving the quality factor and reliability of SPCE, thereby transforming it into a powerful ultrasensitive analytical technique. − Achieving this would provide distinct opportunities for portable and mobile phone-based detection capabilities catering to the internet-of-things. , …”

Optical Detection and Imaging of Nonfluorescent Matter at the Single-Molecule/Particle Level

“…To date, most of the detection methods at the single-molecule level at room temperature rely on the use of molecules with high fluorescence quantum yield combined with efficient spectral rejection of background light. , However, for molecules or materials without fluorescence or with low quantum yield, we cannot use fluorescence microscopy, which strongly limits its application in chemistry and materials science. In addition, fluorescent molecules always suffer other disadvantages, including photobleaching and blinking . To overcome these limitations of fluorescence-based microscopy, many other methods have been developed to detect and image individual nano-objects or molecules.…”

“…In addition, fluorescent molecules always suffer other disadvantages, including photobleaching and blinking. 5 To overcome these limitations of fluorescence-based microscopy, many other methods have been developed to detect and image individual nano-objects or molecules.…”