Functional Plasmonic Microscope: Characterizing the Metabolic Activity of Single Cells via Sub-nm Membrane Fluctuations.

Metabolic abnormalities are at the center of many diseases, and the capability to film and quantify the metabolic activities of a single cell is important for understanding the heterogeneities in these abnormalities. In this paper, a functional plasmonic microscope (FPM) is used to image and measure metabolic activities without fluorescent labels at a single-cell level. The FPM can accurately image and quantify the subnanometer membrane fluctuations with a spatial resolution of 0.5 µm in real time. These active cell membrane fluctuations are caused by metabolic activities across the cell membrane. A three-dimensional (3D) morphology of the bottom cell membrane was imaged and reconstructed with FPM to illustrate the capability of the microscope for cell membrane characterization. Then, the subnanometer cell membrane fluctuations of single cells were imaged and quantified with the FPM using HeLa cells. Cell metabolic heterogeneity is analyzed based on membrane fluctuations of each individual cell that is exposed to similar environmental conditions. In addition, we demonstrated that the FPM could be used to evaluate the therapeutic responses of metabolic inhibitors (glycolysis pathway inhibitor STF 31) on a single-cell level. The result showed that the metabolic activities significantly decrease over time, but the nature of this response varies, depicting cell heterogeneity. A low-concentration dose showed a reduced fluctuation frequency with consistent fluctuation amplitudes, while the high-concentration dose showcased a decreasing trend in both cases. These results have demonstrated the capabilities of the functional plasmonic microscope to measure and quantify metabolic activities for drug discovery.

Exploring the synergy of radiative coupling and substrate undercut in arrayed gold nanodisks for economical, ultra-sensitive label-free biosensing.

We report radiatively coupled arrayed gold nanodisks on invisible substrate (AGNIS) as a cost-effective, high-performance platform for nanoplasmonic biosensing. By substrate undercut, the electric field distribution around the nanodisks has been restored to as if the nanodisks were surrounded by a single medium, thereby provides analyte accessibility to otherwise buried enhanced electric field. The AGNIS substrate has been fabricated by wafer-scale nanosphere lithography without the need for costly lithography. The LSPR blue-shifting behavior synergistically contributed by radiative coupling and substrate undercut have been investigated for the first time, which culminates in a remarkable refractive index sensitivity increase from 207 nm/RIU to 578 nm/RIU. The synergy also improves surface sensitivity to monolayer neutravidin-biotin binding from 7.4 nm to 20.3 nm with the limit of detection (LOD) of neutravidin at 50 fM, which is among the best label-free results reported to date on this specific surface binding reaction. As a potential cancer diagnostic application, extracellular vesicles such as exosomes excreted by cancer and normal cells were measured with a LOD within 112-600 (exosomes/µL), which would be sufficient in many clinical applications. Using CD9, CD63, and CD81 antibodies, label-free profiling has shown increased expression of all three surface antigens in cancer-derived exosomes. This work demonstrates, for the first time, strong synergy of arrayed radiative coupling and substrate undercut can enable economical, ultrasensitive biosensing in the visible light spectrum where high-quality, low-cost silicon detectors are readily available for point-of-care applications.

Investigation of Thermal Properties of Graphene-Coated Membranes by Laser Irradiation to Remove Biofoulants.

In the present study, we take advantage of the high thermal conductivity of graphene nanomaterials to develop a filter that can be easily cleaned via laser irradiation after biofouling occurs. In this investigation, the intensity of the laser beam and the amount of graphene used for membrane coating were investigated with Bacillus subtilis to achieve the most efficient removal of biofoulants. Thermographic measurements of glass microfiber filters coated with 500 µg of graphene showed an increase in temperature of about 328 ± 9 °C in about 6 s when the filters were irradiated with a 21.6 W/cm-2 laser intensity, which allowed successful removal of biofoulants. The thermal cleaning was effective for at least four filtrations without impacting the subsequent microbial removals, which were of ∼5 log for each filtration step followed by laser irradiation. Additionally, the permeability of the coated filters only dropped from 17.8 to 15.9 L/m2s after the laser cleaning procedure. The cleaning procedure was validated by using bayou water with a complex composition of biofoulants. Graphene-coated membranes coupled with laser irradiation afford a very fast and nonhazardous approach to clean biofoulants on graphene-coated membranes.

Integrated Nanogap Platform for Sub-Volt Dielectrophoretic Trapping and Real-Time Raman Imaging of Biological Nanoparticles.

A rapid, label-free, and broadly applicable chemical analysis platform for nanovesicles and subcellular components is highly desirable for diagnostic assays. We demonstrate an integrated nanogap plasmonic sensing platform that combines subvolt dielectrophoresis (DEP) trapping, gold nanoparticles (AuNPs), and a lineated illumination scheme for real-time, surface-enhanced Raman spectroscopy (SERS) imaging of biological nanoparticles. Our system is capable of isolating suspended sub-100 nm vesicles and imaging the Raman spectra of their cargo within seconds, 100 times faster than conventional point-scan Raman systems. Bare AuNPs are spiked into solution and simultaneously trapped with the nanovesicles along the gap to boost local optical fields. In addition, our platform offers simultaneous and delay-free spatial and temporal multiplexing functionality. These nanogap devices can be mass-produced via atomic layer lithography and provide a practical platform for high-speed SERS analysis of biological nanoparticles.

Smartphone Nanocolorimetry for On-Demand Lead Detection and Quantitation in Drinking Water.

Lead ions (Pb2+) contamination in drinking water, a major source of lead poisoning to the general population, is typically detected by bulky and costly laboratory analytical instrument. A mobile analytical device for rapid Pb2+ sensing is a growing demand. Herein, we report smartphone nanocolorimetry (SNC) as a new technique to detect and quantify dissolved Pb2+ in drinking water. Specifically, we have employed a single-step sedimentation approach by mixing a controlled quantity of chromate ion (CrO42-) to react with Pb2+ containing solutions to form highly insoluble lead chromate (PbCrO4) nanoparticles as vivid yellow precipitates. This is followed by microscopic color detection and intensity quantitation at nanoscale level using dark-field smartphone microscopy. The sum of the intensity of yellow pixels bears a highly reproducible relationship with Pb2+ concentration between 1.37 and 175 ppb in deionized water and 5-175 ppb in city tap water. In contrast to traditional colorimetric techniques analyzing bulk color changes, SNC achieves unparalleled sensitivity by combining nanocolorimetry with dark-field microscopy and mobilized the metal ions detection by integrating the detection into the smartphone microscope platform. SNC is rapid and low-cost and has the potential to enable individual citizens to examine Pb2+ content in drinking water on-demand in virtually any environmental setting.

Photothermal generation of programmable microbubble array on nanoporous gold disks.

We present a novel technique to generate microbubbles photothermally by continuous-wave laser irradiation of nanoporous gold disk (NPGD) array covered microfluidic channels. When a single laser spot is focused on the NPGDs, a microbubble can be generated with controlled size by adjusting the laser power. The dynamics of both bubble growth and shrinkage are studied. Using computer-generated holography on a spatial light modulator (SLM), simultaneous generation of multiple microbubbles at arbitrary locations with independent control is demonstrated. A potential application of flow manipulation is demonstrated using a microfluidic X-shaped junction. The advantages of this technique are flexible bubble generation locations, long bubble lifetimes, no need for light-adsorbing dyes, high controllability over bubble size, and relatively lower power consumption.

Nanoporous metals by alloy corrosion: Bioanalytical and biomedical applications.

Nanoporous metals obtained by dealloying have attracted significant attention for their unusual catalytic properties, and as model materials for fundamental studies of structure-property relationships in a variety of research areas. There has been a recent surge in the use of these metals for biomedical and bioanalytical applications, where many exciting opportunities exist. The goal of this article is to provide a review of recent progress in using nanoporous metals for biological applications, including as biosensors for detecting biomarkers of disease and multifunctional neural interfaces for monitoring and modulating the activity of neural tissue. The article emphasizes the unique properties of nanoporous gold and concludes by discussing its utility in addressing important challenges in biomedical devices.

Compressed sensing hyperspectral imaging in the 0.9-2.5 µm shortwave infrared wavelength range using a digital micromirror device and InGaAs linear array detector.

A hyperspectral imaging system based on compressed sensing has been developed to image in the 0.9-2.5 µm shortwave infrared wavelengths. With a programmable digital micromirror device utilized as spatial light modulator, we have successfully performed spectrally resolved image reconstruction with a 256-element InGaAs linear array detector without traditional raster scanning or a push-broom mechanism by a compressed sensing (CS) single-pixel camera approach. The chemical sensitivity of the imaging sensor to near-infrared (NIR) overtone signatures of hydrocarbons was demonstrated using hydrocarbon and ink patterns on glass, showing spectral selectivity for the chemical components. Compared to point-by-point raster scanning, we show that the CS scheme can effectively accelerate image acquisition with lower but reasonable quality.

Modeling the surface of fast-cured polymer droplet lenses for precision fabrication.

Optical lenses with diameter in the millimeter range have found important commercial use in smartphone cameras. Although these lenses are typically made by molding, recent demonstration of fast-cured polymer droplets by inkjet printing has gained interest for cost-effective smartphone microscopy. In this technique, the surface of a fast-cured polydimethylsiloxane droplet obtains dynamic equilibrium via the interplay of surface tension, gravity, thermalization, and a steep viscosity hike. The nature of surface formation involves multiple physical and chemical domains, which represent significant challenges in modeling with the Young-Laplace theory, assuming constant surface tension and viscosity. To overcome these challenges, we introduce the concept of effective surface tension, which allows fast-cured polymer droplets to be modeled as normal liquid droplets with constant viscosity.

Effective Light Directed Assembly of Building Blocks with Microscale Control.

Light-directed forces have been widely used to pattern micro/nanoscale objects with precise control, forming functional assemblies. However, a substantial laser intensity is required to generate sufficient optical gradient forces to move a small object in a certain direction, causing limited throughput for applications. A high-throughput light-directed assembly is demonstrated as a printing technology by introducing gold nanorods to induce thermal convection flows that move microparticles (diameter = 40 µm to several hundreds of micrometers) to specific light-guided locations, forming desired patterns. With the advantage of effective light-directed assembly, the microfluidic-fabricated monodispersed biocompatible microparticles are used as building blocks to construct a structured assembly (≈10 cm scale) in ≈2 min. The control with microscale precision is approached by changing the size of the laser light spot. After crosslinking assembly of building blocks, a novel soft material with wanted pattern is approached. To demonstrate its application, the mesenchymal stem-cell-seeded hydrogel microparticles are prepared as functional building blocks to construct scaffold-free tissues with desired structures. This light-directed fabrication method can be applied to integrate different building units, enabling the bottom-up formation of materials with precise control over their internal structure for bioprinting, tissue engineering, and advanced manufacturing.

Fabrication of multipoint side-firing optical fiber by laser micro-ablation.

A multipoint, side-firing design enables an optical fiber to output light at multiple desired locations along the fiber body. This provides advantages over traditional end-to-end fibers, especially in applications requiring fiber bundles such as brain stimulation or remote sensing. This Letter demonstrates that continuous wave (CW) laser micro-ablation can controllably create conical-shaped cavities, or side windows, for outputting light. The dimensions of these cavities determine the amount of firing light and their firing angle. Experimental data show that a single side window on a 730 µm fiber can deliver more than 8% of the input light. This can be increased to more than 19% on a 65 µm fiber with side windows created using femtosecond laser ablation and chemical etching. Fine control of light distribution along an optical fiber is critical for various biomedical applications such as light-activated drug-release and optogenetics studies.

Automated batch characterization of inkjet-printed elastomer lenses using a LEGO platform.

Small, self-adhesive, inkjet-printed elastomer lenses have enabled smartphone cameras to image and resolve microscopic objects. However, the performance of different lenses within a batch is affected by hard-to-control environmental variables. We present a cost-effective platform to perform automated batch characterization of 300 lens units simultaneously for quality inspection. The system was designed and configured with LEGO bricks, 3D printed parts, and a digital camera. The scheme presented here may become the basis of a high-throughput, in-line inspection tool for quality control purposes and can also be employed for optimization of the manufacturing process.

Nanoporous Gold Nanocomposites as a Versatile Platform for Plasmonic Engineering and Sensing.

Plasmonic metal nanostructures have shown great potential in sensing applications. Among various materials and structures, monolithic nanoporous gold disks (NPGD) have several unique features such as three-dimensional (3D) porous network, large surface area, tunable plasmonic resonance, high-density hot-spots, and excellent architectural integrity and environmental stability. They exhibit a great potential in surface-enhanced spectroscopy, photothermal conversion, and plasmonic sensing. In this work, interactions between smaller colloidal gold nanoparticles (AuNP) and individual NPGDs are studied. Specifically, colloidal gold nanoparticles with different sizes are loaded onto NPGD substrates to form NPG hybrid nanocomposites with tunable plasmonic resonance peaks in the near-infrared spectral range. Newly formed plasmonic hot-spots due to the coupling between individual nanoparticles and NPG disk have been identified in the nanocomposites, which have been experimentally studied using extinction and surface-enhanced Raman scattering. Numerical modeling and simulations have been employed to further unravel various coupling scenarios between AuNP and NPGDs.

Simultaneous Chemical and Refractive Index Sensing in the 1-2.5 µm Near-Infrared Wavelength Range on Nanoporous Gold Disks.

Near-infrared (NIR) absorption spectroscopy provides molecular and chemical information based on overtones and combination bands of the fundamental vibrational modes in the infrared wavelengths. However, the sensitivity of NIR absorption measurement is limited by the generally weak absorption and the relatively poor detector performance compared to other wavelength ranges. To overcome these barriers, we have developed a novel technique to simultaneously obtain chemical and refractive index sensing in 1-2.5 µm NIR wavelength range on nanoporous gold (NPG) disks, which feature high-density plasmonic hot-spots of localized electric field enhancement. For the first time, surface-enhanced near-infrared absorption (SENIRA) spectroscopy has been demonstrated for high sensitivity chemical detection. With a self-assembled monolayer (SAM) of octadecanethiol (ODT), an enhancement factor (EF) of up to ∼10(4) has been demonstrated for the first C-H combination band at 2400 nm using NPG disk with 600 nm diameter. Together with localized surface plasmon resonance (LSPR) extinction spectroscopy, simultaneous sensing of sample refractive index has been achieved for the first time. The performance of this technique has been evaluated using various hydrocarbon compounds and crude oil samples.

Direct-write patterning of nanoporous gold microstructures by in situ laser-assisted dealloying.

We report a novel patterning technique to direct-write microscale nanoporous gold (NPG) features by projecting laser patterns using a spatial light modulator (SLM) onto an Au/Ag alloy film immersed in diluted nitric acid solutions. Heat accumulation induced by the photothermal effect enables localized dealloying in such solutions, which is otherwise impotent at room temperature. Consequently, NPG micropatterns are formed at the irradiated spots while the surrounding alloy remains intact. We have studied the size of the patterned NPG microstructures with respect to laser power and irradiation time. The NPG microstructures become significantly more transparent compared to the original alloy film. The NPG microstructures also exhibit strong localized surface plasmon resonance (LSPR) which is otherwise weak in the original alloy film. Both the light transmission intensity and LSPR peak wavelength have been demonstrated to be sensitive to the local environmental refractive index as quantified by microscopy and spectroscopy.

A sandwich substrate for ultrasensitive and label-free SERS spectroscopic detection of folic acid / methotrexate.

A highly sensitive surface enhanced Raman scattering (SERS) substrate with particle-film sandwich geometry has been developed for the label free detection of folic acid (FA) and methotrexate (MTX). In this sandwich structure, the bottom layer is composed of a copper foil decorated with silver nanoparticles synthesized by the galvanic displacement reaction, and top layer is constituted by silver nanoparticles. The FA and MTX molecules are sandwiched between the silver nanoparticles decorated copper film and the silver nanoparticles. The plasmonic coupling between the two layers of the sandwich structure greatly enhances the SERS spectra of FA and MTX. SERS activity of the substrate was studied and optimized by adjusting the time of galvanic displacement reaction. The SERS spectra of the FA and MTX showed the minimum detection concentration of 100 pM. The identification of methotrexate and folic acid analogs was also carried out by SERS spectra analysis.

Analysis of ethyl and methyl centralite vibrational spectra for mapping organic gunshot residues.

Detection of ethyl and methyl centralites in gunshot residues is important in forensic science due to their limited contamination from environmental sources compared to other organic residues. However, the vibrational frequencies of centralites are little explored and their frequency assignments are incomplete. Herein, we investigated vibrational frequencies of centralites based on Density functional theory (DFT) to understand their vibrations. The simulated frequencies exhibit excellent agreement with the experimental data, and the detailed assignments are comprehensively elaborated. We also demonstrate that centralite particles could be detected through Raman imaging based on their fingerprints. This work is very important for the further vibrational studies in detecting and tracing centralites in gunshot residues.

Performance of line-scan Raman microscopy for high-throughput chemical imaging of cell population.

We evaluate the performance of line-scan Raman microscopy (LSRM), a versatile label-free technique, for high-throughput chemical imaging of cell population. We provide detailed design and configuration of a home-built LSRM system developed in our laboratory. By exploiting parallel acquisition, the LSRM system achieves a significant throughput advantage over conventional point-scan Raman microscopy by projecting a laser line onto the sample and imaging the Raman scattered light from the entire line using a grating spectrograph and a charge-coupled device (CCD) camera. Two-dimensional chemical maps can be generated by scanning the projected line in the transverse direction. The resolution in the x and y direction has been characterized to be ~600-800 nm for 785 nm laser excitation. Our system enables rapid classification of microparticles with similar shape, size, and refractive index based on their chemical composition. An equivalent imaging throughput of 100 microparticles/s for 1 µm polystyrene beads has been achieved. We demonstrate the application of LSRM to imaging bacterial spores by identifying endogenous calcium dipicolinate. We also demonstrate that LSRM enables the study of intact microalgal cells at the colonial level and the identification of intra- and extracellular chemical constituents and metabolites, such as chlorophyll, carotenoids, lipids, and hydrocarbons. We conclude that LSRM can be an effective and practical tool for obtaining endogenous microscopic chemical and molecular information from cell population.

Plasmonic nano-aperture label-free imaging of single small extracellular vesicles for cancer detection.

BACKGROUND: Small extracellular vesicle (sEV) analysis can potentially improve cancer detection and diagnostics. However, this potential has been constrained by insufficient sensitivity, dynamic range, and the need for complex labeling. METHODS: In this study, we demonstrate the combination of PANORAMA and fluorescence imaging for single sEV analysis. The co-acquisition of PANORAMA and fluorescence images enables label-free visualization, enumeration, size determination, and enables detection of cargo microRNAs (miRs). RESULTS: An increased sEV count is observed in human plasma samples from patients with cancer, regardless of cancer type. The cargo miR-21 provides molecular specificity within the same sEV population at the single unit level, which pinpoints the sEVs subset of cancer origin. Using cancer cells-implanted animals, cancer-specific sEVs from 20 µl of plasma can be detected before tumors were palpable. The level plateaus between 5-15 absolute sEV count (ASC) per µl with tumors ≥8 mm3. In healthy human individuals (N = 106), the levels are on average 1.5 ASC/µl (+/- 0.95) without miR-21 expression. However, for stage I-III cancer patients (N = 205), nearly all (204 out of 205) have levels exceeding 3.5 ASC/µl with an average of 12.2 ASC/µl (±9.6), and a variable proportion of miR-21 labeling among different tumor types with 100% cancer specificity. Using a threshold of 3.5 ASC/µl to test a separate sample set in a blinded fashion yields accurate classification of healthy individuals from cancer patients. CONCLUSIONS: Our techniques and findings can impact the understanding of cancer biology and the development of new cancer detection and diagnostic technologies.

Small extracellular vesicles (sEVs) are tiny particles derived from cells that can be detected in bodily fluids such as blood. Detecting sEVs and analyzing their contents may potentially help us to diagnose disease, for example by observing differences in sEV numbers or contents in the blood of patients with cancer versus healthy people. Here, we combine two imaging methods ­ our previously developed method PANORAMA and imaging of fluorescence emitted by sEVs­to visualize and count sEVs, determine their size, and analyze their cargo. We observe differences in sEV numbers and cargo in samples taken from healthy people versus people with cancer and are able to differentiate these two populations based on our analysis of sEVs. With further testing, our approach may be a useful tool for cancer diagnosis and provide insights into the biology of cancer and sEVs.

Single-Exosome Counting and 3D, Subdiffraction Limit Localization Using Dynamic Plasmonic Nanoaperture Label-Free Imaging.

Blood-circulating exosomes as a disease biomarker have great potential in clinical applications as they contain molecular information about their parental cells. However, label-free characterization of exosomes is challenging due to their small size. Without labeling, exosomes are virtually indistinguishable from other entities of similar size. Over recent years, several techniques have been developed to overcome the existing challenges. This paper demonstrates a new label-free approach based on dynamic PlAsmonic NanO-apeRture lAbel-free iMAging (D-PANORAMA), a bright-field technique implemented on arrayed gold nanodisks on invisible substrates (AGNIS). PANORAMA provides high surface sensitivity and has been shown to count single 25 nm polystyrene beads (PSB) previously. Herein, we show that using the dynamic imaging mode, D-PANORAMA can yield 3-dimensional, sub-diffraction limited localization of individual 25 nm beads. Furthermore, we demonstrate D-PANORAMA's capability to size, count, and localize the 3-dimensional, sub-diffraction limited position of individual exosomes as they bind to the AGNIS surface. We emphasize the importance of both the in-plane and out-of-plane localization, which exploit the synergy of 2-dimensional imaging and the intensity contrast.