Defocused imaging-based quantification of plasmon-induced distortion of single emitter emission.

Optical properties of single emitters can be significantly improved through the interaction with plasmonic structures, leading to enhanced sensing and imaging capabilities. In turn, single emitters can act as sensitive probes of the local electromagnetic field surrounding plasmonic structures, furnishing fundamental insights into their physics and guiding the design of novel plasmonic devices. However, the interaction of emitters in the proximity to a plasmonic nanostructure causes distortion, which hinders precise estimation of position and polarization state and is one of the reasons why detection and quantification of molecular processes yet remain fundamentally challenging in this era of super-resolution. Here, we investigate axially defocused images of a single fluorescent emitter near metallic nanostructure, which encode emitter positions and can be acquired in the far-field with high sensitivity, while analyzing the images with pattern matching algorithm to explore emitter-localized surface plasmon interaction and retrieve information regarding emitter positions. Significant distortion in defocused images of fluorescent beads and quantum dots near nanostructure was observed and analyzed by pattern matching and finite-difference time-domain methods, which revealed that the distortion arises from the emitter interaction with nanostructure. Pattern matching algorithm was also adopted to estimate the lateral positions of a dipole that models an emitter utilizing the distorted defocused images and achieved improvement by more than 3 times over conventional diffraction-limited localization methods. The improvement by defocused imaging is expected to provide a way of enhancing reliability when using plasmonic nanostructure and diversifying strategies for various imaging and sensing modalities.

CRISPR/Cas13a-Based MicroRNA Detection in Tumor-Derived Extracellular Vesicles.

MicroRNAs (miRNAs) in extracellular vesicles (EVs) play essential roles in cancer initiation and progression. Quantitative measurements of EV miRNAs are critical for cancer diagnosis and longitudinal monitoring. Traditional PCR-based methods, however, require multi-step procedures and remain as bulk analysis. Here, the authors introduce an amplification-free and extraction-free EV miRNA detection method using a CRISPR/Cas13a sensing system. CRISPR/Cas13a sensing components are encapsulated in liposomes and delivered them into EVs through liposome-EV fusion. This allows for accurately quantify specific miRNA-positive EV counts using 1 × 108  EVs. The authors show that miR-21-5p-positive EV counts are in the range of 2%-10% in ovarian cancer EVs, which is significantly higher than the positive EV counts from the benign cells (<0.65%). The result show an excellent correlation between bulk analysis with the gold-standard method, RT-qPCR. The authors also demonstrate multiplexed protein-miRNA analysis in tumor-derived EVs by capturing EpCAM-positive EVs and quantifying miR-21-5p-positive ones in the subpopulation, which show significantly higher counts in the plasma of cancer patients than healthy controls. The developed EV miRNA sensing system provides the specific miRNA detection method in intact EVs without RNA extraction and opens up the possibility of multiplexed single EV analysis for protein and RNA markers.

Electrokinetically enhanced label-free plasmonic sensing for rapid detection of tumor-derived extracellular vesicles.

ANALYSIS: of rare circulating extracellular vesicles (EV) from early cancers or different types of host cells requires extremely sensitive EV sensing technologies. Nanoplasmonic EV sensing technologies have demonstrated good analytical performances, but their sensitivity is often limited by EVs' diffusion to the active sensor surface for specific target EV capture. Here, we developed an advanced plasmonic EV platform with electrokinetically enhanced yields (KeyPLEX). The KeyPLEX system effectively overcomes diffusion-limited reactions with applied electroosmosis and dielectrophoresis forces. These forces bring EVs toward the sensor surface and concentrate them in specific areas. Using the keyPLEX, we showed significant improvements in detection sensitivity by ∼100-fold, leading to the sensitive detection of rare cancer EVs from human plasma samples in 10 min. The keyPLEX system could become a valuable tool for point-of-care rapid EV analysis.

Plasmon-Enhanced Characterization of Single Extracellular Vesicles.

Extracellular vesicles (EVs) represent heterogeneous populations of membrane-bound vesicles shed from almost all kinds of cells. Although superior to conventional methods, most newly developed EV sensing platforms still require a certain number of EVs, measuring bulk signals from a group of vesicles. A new analytical approach that enables single EV analysis could be extremely valuable for understanding EVs' subtypes, heterogeneity, and production dynamics during disease development and progression. Here, we describe a new nanoplasmonic sensing platform for sensitive single EV analysis. Termed nPLEX-FL (nano-plasmonic EV analysis with enhanced fluorescence detection), the system amplifies EVs' fluorescence signals using periodic gold nanohole structures, enabling sensitive, multiplexed analysis of single EVs.

Rapid PCR kit: lateral flow paper strip with Joule heater for SARS-CoV-2 detection.

Polymerase chain reaction (PCR)-based diagnostic kits for point-of-care (POC) testing are highly desirable to prevent the spread of infectious diseases. Here, we demonstrate a rapid PCR testing kit that involves integrating a lateral flow paper strip with a nichrome-based thin film heater. The use of a paper membrane as a PCR-solution container results in fast thermocycling without a cooler because the membrane can contain the solution with a high specific surface area where Joule heating is applied. After PCR, amplified products are simultaneously detected at the lateral flow paper strip with the naked eye. Severe acute respiratory syndrome ß-coronavirus RNA can be detected within 30 min after PCR solution injection. This work reveals that the paper membrane can act as not only a capillary flow channel but also as a promising platform for fast PCR and detection.

Plasmon-Enhanced Single Extracellular Vesicle Analysis for Cholangiocarcinoma Diagnosis.

Cholangiocarcinoma (CCA) is a fatal disease often detected late in unresectable stages. Currently, there are no effective diagnostic methods or biomarkers to detect CCA early with high confidence. Analysis of tumor-derived extracellular vesicles (tEVs) harvested from liquid biopsies can provide a new opportunity to achieve this goal. Here, an advanced nanoplasmonic sensing technology is reported, termed FLEX (fluorescence-amplified extracellular vesicle sensing technology), for sensitive and robust single EV analysis. In the FLEX assay, EVs are captured on a plasmonic gold nanowell surface and immunolabeled for cancer-associated biomarkers to identify tEVs. The underlying plasmonic gold nanowell structures then amplify EVs' fluorescence signals, an effective amplification process at the single EV level. The FLEX EV analysis revealed a wide heterogeneity of tEVs and their marker levels. FLEX also detected small tEVs not detected by conventional EV fluorescence imaging due to weak signals. Tumor markers (MUC1, EGFR, and EPCAM) are identified in CCA, and this marker combination is applied to detect tEVs in clinical bile samples. The FLEX assay detected CCA with an area under the curve of 0.93, significantly better than current clinical markers. The sensitive and accurate nanoplasmonic EV sensing technology can aid in early CCA diagnosis.

Physisorption of Affinity Ligands Facilitates Extracellular Vesicle Detection with Low Non-Specific Binding to Plasmonic Gold Substrates.

Plasmonic biosensors are increasingly being used for the analysis of extracellular vesicles (EVs) originating from disease areas. However, the high non-specific binding of EVs to a gold-sensing surface has been a critical problem and hindered the true translational potential. Here, we report that direct antibody immobilization on the plasmonic gold surface via physisorption shows excellent capture of cancer-derived EVs with ultralow non-specific binding even at very high concentrations. Contrary to commonly used methods that involve thiol-based linker attachment and an EDC/sulfo-NHS reaction, we show a higher specific capture rate and >50-fold lower non-specific on citrate-capped plain and nanopatterned gold surfaces. The method provides a simple, fast, and reproducible means to functionalize plasmonic gold surfaces with antibodies for robust EV biosensing.

Plasmon-enhanced fluorescence correlation spectroscopy for super-localized detection of nanoscale subcellular dynamics.

In this report, we investigate plasmon-enhanced imaging fluorescence correlation spectroscopy (p-FCS). p-FCS takes advantage of extreme light confinement by localization at nanogap-based plasmonic nanodimer arrays (PNAs) for enhanced signal-to-noise ratio (SNR) and improved precision by registration with surface plasmon microscopy images. Theoretical results corroborate the enhancement by PNAs in the far-field. Near-field scanning optical microscopy was used to confirm near-field localization experimentally. Experimental confirmation was also conducted with fluorescent nanobeads. The concept was further applied to studying the diffusion dynamics of lysosomes in HEK293T cells stimulated by phorbol 12-myristate 13-acetate treatment. It was found that lysosomes demonstrate stronger super-diffusive behavior with relatively weaker sub-diffusion after stimulation. SNR measured of p-FCS was improved by 9.77 times over conventional FCS. This report is expected to serve as the foundation for an enhanced analytical tool to explore subcellular dynamics.

Plasmonic Sensors for Extracellular Vesicle Analysis: From Scientific Development to Translational Research.

Extracellular vesicles (EVs), actively shed from a variety of neoplastic and host cells, are abundant in blood and carry molecular markers from parental cells. For these reasons, EVs have gained much interest as biomarkers of disease. Among a number of different analytical methods that have been developed, surface plasmon resonance (SPR) stands out as one of the ideal techniques given its sensitivity, robustness, and ability to miniaturize. In this Review, we compare different SPR platforms for EV analysis, including conventional SPR, nanoplasmonic sensors, surface-enhanced Raman spectroscopy, and plasmonic-enhanced fluorescence. We discuss different surface chemistries used to capture targeted EVs and molecularly profile their proteins and RNAs. We also highlight these plasmonic platforms' clinical applications, including cancers, neurodegenerative diseases, and cardiovascular diseases. Finally, we discuss the future perspective of plasmonic sensing for EVs and their potentials for commercialization and clinical translation.

Astrocytes Control Sensory Acuity via Tonic Inhibition in the Thalamus.

Sensory discrimination is essential for survival. However, how sensory information is finely controlled in the brain is not well defined. Here, we show that astrocytes control tactile acuity via tonic inhibition in the thalamus. Mechanistically, diamine oxidase (DAO) and the subsequent aldehyde dehydrogenase 1a1 (Aldh1a1) convert putrescine into GABA, which is released via Best1. The GABA from astrocytes inhibits synaptically evoked firing at the lemniscal synapses to fine-tune the dynamic range of the stimulation-response relationship, the precision of spike timing, and tactile discrimination. Our findings reveal a novel role of astrocytes in the control of sensory acuity through tonic GABA release.

Plasmon-Enhanced Biosensing for Multiplexed Profiling of Extracellular Vesicles.

Extracellular vesicles (EVs)-nanoscale phospholipid vesicles secreted by cells-present new opportunities for molecular diagnosis from non-invasive liquid biopsies. Single EV protein analysis can be extremely valuable in studying EVs as circulating cancer biomarkers, but it is technically challenging due to weak detection signals associated with limited amounts of epitopes and small surface areas for antibody labeling. Here, a new, simple method that enables multiplexed analyses of EV markers with improved sensitivities is reported. Specifically, plasmon-enhanced fluorescence detection is implemented that amplifies fluorescence signals using surface plasmon resonances excited by periodic gold nanohole structures. It is shown that fluorescence signals in multiple channels are amplified by one order of magnitude, and both transmembrane and intravesicular markers can be detected at the single EV level. This approach can offer additional insight into understanding subtypes, heterogeneity, and production dynamics of EVs during disease development and progression.

Enhanced surface plasmon microscopy based on multi-channel spatial light switching for label-free neuronal imaging.

In this paper, we have investigated multi-channel switching of light incidence in multiple directions to improve image clarity in surface plasmon microscopy (SPM) for robust and consistent imaging performance regardless of the pattern geometry and shape. Multi-channel light switching in SPM allows significant reduction of adverse scattering effects by surface plasmon (SP). For proof of concept, an eight-channel spatially switched SPM (ssSPM) system has been set up. The results with reference objects including square arrays and Siemens stars experimentally confirm much improved images with ssSPM by reducing the artifacts of SP scattering significantly. On a quantitative basis, contrast analysis preformed with square arrays shows image contrast enhanced by more than three times over conventional SPM. Three image reconstruction algorithms were evaluated for optimal image acquisition. It is suggested that averaging combined with minimum-filtering produces the highest resolution. ssSPM was applied to label-free imaging of primary neuron cultures and shown to present enhanced images with clarity far better than conventional SPM.

Deep Learning Approach for Enhanced Detection of Surface Plasmon Scattering.

A deep learning approach has been taken to improve detection characteristics of surface plasmon microscopy (SPM) of light scattering. Deep learning based on the convolutional neural network algorithm was used to estimate the effect of scattering parameters, mainly the number of scatterers. The improvement was assessed on a quantitative basis by applying the approach to SPM images formed by coherent interference of scatterers. It was found that deep learning significantly improves the accuracy over conventional detection: the enhancement in the accuracy was shown to be significantly higher by almost 6 times and useful for scattering by polydisperse mixtures. This suggests that deep learning can be used to find scattering objects effectively in the noisy environment. Furthermore, deep learning can be extended directly to label-free molecular detection assays and provide considerably improved detection in imaging and microscopy techniques.

Superlocalized Three-Dimensional Live Imaging of Mitochondrial Dynamics in Neurons Using Plasmonic Nanohole Arrays.

We investigated the transport of neuronal mitochondria using superlocalized near-fields with plasmonic nanohole arrays (PNAs). Compared to traditional imaging techniques, PNAs create a massive array of superlocalized light beams and allow 3D mitochondrial dynamics to be sampled and extracted almost in real time. In this work, mitochondrial fluorescence excited by the PNAs was captured by an optical microscope using dual objective lenses, which produced superlocalized dynamics while minimizing light scattering by the plasmonic substrate. It was found that mitochondria move with an average velocity 0.33 ± 0.26 µm/s, a significant part of which, by almost 50%, was contributed by the movement along the depth axis ( z-axis). Mitochondrial positions were acquired with superlocalized precision (σ x = 5.7 nm and σ y = 11.8 nm) in the lateral plane and σ z = 78.7 nm in the z-axis, which presents an enhancement by 12.7-fold in resolution compared to confocal fluorescence microscopy. The approach is expected to serve as a way to provide 3D information on molecular dynamics in real time.

Surface plasmon microscopy by spatial light switching for label-free imaging with enhanced resolution.

In this Letter, we describe spatially switched surface plasmon microscopy (ssSPM) based on two-channel momentum sampling. The performance evaluated with periodic nanowires in comparison with conventional SPM and bright-field microscopy shows that the resolution of ssSPM is enhanced by almost 15 times over conventional SPM. ssSPM provides an extremely simple way to attain diffraction limit in SPM and to go beyond for super-resolution in label-free microscopy techniques.

Molecular overlap with optical near-fields based on plasmonic nanolithography for ultrasensitive label-free detection by light-matter colocalization.

In this work, we investigate the detection sensitivity of surface plasmon resonance (SPR) biosensors by engineering spatial distribution of electromagnetic near-fields for colocalization with molecular distribution. The light-matter colocalization was based on plasmonic nanolithography, the concept of which was confirmed by detecting streptavidin biotin interactions on triangular nanoaperture arrays after the structure of the aperture arrays was optimized for colocalization efficiency. The colocalization was shown to amplify optical signature significantly and thereby to achieve detection on the order of 100 streptavidin molecules with a binding capacity below 1fg/mm2, an enhancement by more than three orders of magnitude over conventional SPR detection.

Ultra-Sensitive Surface Plasmon Resonance Detection by Colocalized 3D Plasmonic Nanogap Arrays.

Ultra-sensitive detection based on surface plasmon resonance (SPR) was investigated using 3D nanogap arrays for colocalization of target molecular distribution and localized plasmon wave in the near-field. Colocalization was performed by oblique deposition of a dielectric mask layer to create nanogap at the side of circular and triangular nanoaperture, where fields localized by surface plasmon localization coincide with the spatial distribution of target molecular interactions. The feasibility of ultra-sensitivity was experimentally verified by measuring DNA hybridization. Triangular nanopattern provided an optimum to achieve highly amplified angular shifts and led to enhanced detection sensitivity on the order of 1 fg/mm2 in terms of molecular binding capacity. We confirmed improvement of SPR sensitivity by three orders of magnitude, compared with conventional SPR sensors, using 3D plasmonic nanogap arrays.

Localization-based full-field microscopy: how to attain super-resolved images.

In this study, we have investigated localization-based microscopy to achieve full-field super-resolution. For localized sampling, we have considered combs consisting of unit pulses and near-fields localized by surface nanoapertures. Achievable images after reconstruction were assessed in terms of peak signal-to-noise ratio (PSNR). It was found that spatial switching of individual pulses may be needed to break the diffraction limit. Among the parameters, the resolution was largely determined by sampling period while the effect of width of a sampling pulse on PSNR was relatively limited. For the range of sampling parameters that we considered, the highest resolution achievable is estimated to be 70 nm, which can further be enhanced by optimizing the localization parameters.

Surface plasmon-enhanced nanoscopy of intracellular cytoskeletal actin filaments using random nanodot arrays.

The feasibility of super-resolution microscopy has been investigated based on random localization of surface plasmon using blocked random nanodot arrays. The resolution is mainly determined by the size of localized fields in the range of 100-150 nm. The concept was validated by imaging FITC-conjugated phalloidin that binds to cellular actin filaments. The experimental results confirm improved resolution in reconstructed images. Effect of far-field registration on image reconstruction was also analyzed. Correlation between reconstructed images was maintained to be above 81% after registration. Nanodot arrays are synthesized by temperature-annealing without sophisticated lithography and thus can be mass-produced in an extremely large substrate. The results suggest a super-resolution imaging technique that can be accessible and available in large amounts.

Revolutionizing the FRET-based light emission in core-shell nanostructures via comprehensive activity of surface plasmons.

We demonstrate the surface-plasmon-induced enhancement of Förster resonance energy transfer (FRET)using a model multilayer core-shell nanostructure consisting of an Au core and surrounding FRET pairs, i.e., CdSe quantum dot donors and S101 dye acceptors. The multilayer configuration was demonstrated to exhibit synergistic effects of surface plasmon energy transfer from the metal to the CdSe and plasmon-enhanced FRET from the quantum dots to the dye. With precise control over the distance between the components in the nanostructure, significant improvement in the emission of CdSe was achieved by combined resonance energy transfer and near-field enhancement by the metal, as well as subsequent improvement in the emission of dye induced by the enhanced emission of CdSe. Consequently, the Förster radius was increased to 7.92 nm and the FRET efficiency was improved to 86.57% in the tailored plasmonic FRET nanostructure compared to the conventional FRET system (22.46%) without plasmonic metals.