Understanding the Origin of Surface Plasmon Resonance Microscopy Signals in Cell-Based Molecular Interaction Measurements.

Surface plasmon resonance microscopy (SPRM) has been massively applied for near-field optical measurement, sensing, and imaging because of its high detection sensitivity, nondestructive, noninvasive, wide-field, and label-free imaging capabilities. However, the transverse propagation characteristic of the surface plasmon wave generated during surface plasmon resonance (SPR) leads to notable “tail” patterns in the SPR image, which severely deteriorates the image quality. Here, we propose an incidence angle scanning method in SPRM to obtain a resonance angle image with exceptional contrast that significantly mitigates the adverse effects of “tail” patterns. The resonance angle image provides the complete morphology of the analyzed samples and enables two-dimensional quantification, which is incapable in conventional SPRM. The effectiveness of the method was experimentally verified using photoresist square samples with different sizes and two-dimensional materials with various geometric shapes. The edges of samples were fully reconstructed and a maximum fivefold increase in the image contrast has been achieved. Our method offers a convenient way to enhance the SPRM imaging capabilities with low cost and stable performance, which greatly expands the applications of SPRM in label-free detection, imaging, and quantification.

Studying the electrochemistry of single nanoparticles with surface plasmon resonance microscopy

Surface plasmon resonance microscopy (SPRM) is a powerful label-free imaging technique with spatial resolution approaching the optical diffraction limit. The high sensitivity of SPRM to small changes in index of refraction at an interface allows imaging of dynamic protein structures within a cell. Visualization of subcellular features, such as focal adhesions (FAs), can be performed on live cells using a high numerical aperture objective lens with a digital light projector to precisely position the incident angle of the excitation light. Within the cell-substrate region of the SPRM image, punctate regions of high contrast are putatively identified as the cellular FAs. Optical parameter analysis is achieved by application of the Fresnel model to the SPRM data and resulting refractive index measurements are used to calculate protein density and mass. FAs are known to be regions of high protein density that reside at the cell-substratum interface. Comparing SPRM with fluorescence images of antibody stained for vinculin, a component in FAs, reveals similar measurements of FA size. In addition, a positive correlation between FA size and protein density is revealed by SPRM. Comparing SPRM images for two cell types reveals a distinct difference in the protein density and mass of their respective FAs. Application of SPRM to quantify mass can greatly aid monitoring basic processes that control FA mass and growth and contribute to accurate models that describe cell-extracellular interactions.

Cell-based kinetic studies of ligand or candidate drug binding to membrane proteins have produced affinity and kinetic values that are different from measurements using purified proteins. However, ligand binding to fixated cells whose membrane constituents (e.g., proteins and their glycosylated forms) are partially connected by a cross-linking reagent has not been compared to that to live cells. Under the same experimental conditions for the LigandTracer method, we measured the interactions of fluorophore-labeled lectins and antibody molecules with glycans at HFF cells and the human epithelial growth receptor 2 at SKBR3 cells, respectively. In conjunction with surface plasmon resonance microscopy, the effects of labels and cell/sub-cell heterogeneity on binding kinetics were investigated. Our results revealed that, for cell constituents whose structures and functions are not closely dependent on cell viability, the ligand binding kinetics at fixated cells is only slightly different from that at live cells. The altered kinetics is explained on the basis of a less mobile receptor confined in a local environment created by partially interconnected protein molecules. We show that cell/sub-cell heterogeneity and labels on the ligands can alter the binding reaction more significantly. Thus, fixating cells not only simplifies experimental procedures for drug screening and renders assays more robust but also provides reliable kinetic information about drug binding to cell constituents whose structures are not changed by chemical fixation.

Benefitting from high sensitivity, real-time, and label-free imaging, surface plasmon resonance microscopy (SPRM) has become a powerful tool for dynamic detection of nanoparticles. However, the evanescent propagation of surface plasmon polaritons (SPPs) induces interference between scattered and launched SPPs, which deteriorates the spatial resolution and signal-to-noise ratio (SNR). Due to the simplicity and fast processing, image reconstruction based on deconvolution has shown the feasibility of improving the spatial resolution of SPRM imaging. Retrieving the particle scattering from SPRM interference imaging by filters is crucial for reconstruction. In this work, we illustrate the effect of filters extracting SPP scattering of nanoparticles with different sizes and shapes for reconstruction. The results indicate that the optimum filters are determined by the material of nanoparticles instead of particle sizes. The reconstruction of single Au and PS nanospheres as well as Ag nanowires with optimum filters is achieved. The reconstructed spatial resolution is improved to 254 nm, and the SNR is increased by 8.1 times. Our research improves the quality of SPRM imaging and provides a reliable method for fast detection of particles with diverse sizes and shapes.

Surface plasmon resonance microscopy (SPRM) combines the principles of traditional microscopy with the versatility of surface plasmons to develop label-free imaging methods. This paper describes a proof-of-principles approach based on deep learning that utilized the Y-Net convolutional neural network model to improve the detection and analysis methodology of SPRM. A machine-learning based image analysis technique was used to provide a method for the one-shot analysis of SPRM images to estimate scattering parameters such as the scatterer location. The method was assessed by applying the approach to SPRM images and reconstructing an image from the network output for comparison with the original image. The results showed that deep learning can localize scatterers and predict other variables of scattering objects with high accuracy in a noisy environment. The results also confirmed that with a larger field of view, deep learning can be used to improve traditional SPRM such that it localizes and produces scatterer characteristics in one shot, considerably increasing the detection capabilities of SPRM.

Surface plasmon resonance microscopy (SPRM) is an excellent platform for in situ studying cell-substrate interactions. However, SPRM suffers from poor spatial resolution and small field of view. Herein, we demonstrate plasmonic scattering microscopy (PSM) by adding a dry objective on a popular prism-coupled surface plasmon resonance (SPR) system. PSM not only retains SPRM's high sensitivity and real-time analysis capability, but also provides ≈7 times higher spatial resolution and ≈70 times larger field of view than the typical SPRM, thus providing more details about membrane protein response to ligand binding on over 100 cells simultaneously. In addition, PSM allows quantifying the target movements in the axial direction with a high spatial resolution, thus allowing mapping adhesion spring constants for quantitatively describing the mechanical properties of the cell-substrate contacts. This work may offer a powerful and cost-effective strategy for upgrading current SPR products.

Label-free optical imaging and sensing of single nanoparticles are vital for fundamental research, disease diagnosis, and nanomaterial studies. Surface plasmon resonance microscopy (SPRM) is a label-free detection technology which is widely used in the detection of single nanoparticles. However, conventional SPRM suffers from poor spatial resolution, a limited field-of-view, system complexity, and high operating costs. In this study, we introduce a compact, low-cost, and large field-of-view chip-based plasmonic scattering microscopy (Chip-PSM). Compared with SPRM, Chip-PSM retains high detection sensitivity and in situ label-free analysis capability, while offering a larger field-of-view, an isotropic point-spread-function and higher spatial resolution. With these advantages, Chip-PSM enables detecting and imaging dielectric nanoparticles, gold nanoparticles, and biological samples. Additionally, the hygroscopic growth dynamics of aerosol nanoparticles and the chemical reactions occurring on nanocrystals are successfully characterized via Chip-PSM. We anticipate that the proposed Chip-PSM will have broad applications across many scientific fields, including physics, chemistry, and atmospheric sciences.

Gold colloid changes its color when the internanoparticle distance changes. On the basis of analyte-induced aggregation or disaggregation behavior of gold nanoparticles (AuNPs), versatile colorimetric assays have been developed for measuring various kinds of analytes including proteins, DNA, small molecules, and ions. Traditional read-out signals, which are usually measured by a spectrometer or naked eyes, are based on the averaged extinction properties of a bulk solution containing billions of nanoparticles. Averaged extinction property of a large amount of nanoparticles diminished the contribution from rare events when the analyte concentration was low, thus resulting in limited detection sensitivity. Instead of measuring the averaged optical property from bulk colloid, in the present work, we proposed a digital counterpart of the colorimetric assay by imaging and counting individual AuNPs. This method quantified the analyte concentration with the number percentage of large-sized AuNPs aggregates, which were digitally counted with surface plasmon resonance microscopy (SPRM), a plasmonic imaging technique recently developed by us and other groups. SPRM was able to identify rare AuNPs aggregates despite their small population and greatly improved the detection sensitivity as demonstrated by two model systems based on analyte-induced aggregation and disaggregation, respectively. Furthermore, besides plasmonic AuNPs, SPRM is also suitable for imaging and counting nonplasmonic nanomaterials such as silica and metal oxide with poor extinction properties. It is thus anticipated that the present digitized assay holds a great potential for expanding the colorimetric assay to broad categories of nonplasmonic nanoparticles.

Exosome analysis is a promising tool for clinical and biological research applications. However, detection and biomarker quantification of exosomes is technically challenging because they are small and highly heterogeneous. Here, we report an optical approach for imaging exosomes and quantifying their protein markers without labels using plasmonic scattering microscopy (PSM). PSM can provide improved spatial resolution and distortion-free image compared to conventional surface plasmon resonance (SPR) microscopy, with the signal-to-noise ratio similar to objective coupled surface plasmon resonance (SPR) microscopy, and millimeter-scale field of view as a prism-coupled SPR system, thus allowing exosome size distribution analysis with high throughput. In addition, PSM retains the high specificity and surface sensitivity of the SPR sensors and thus allows selection of exosomes from extracellular vesicles with antibody-modified sensor surfaces and in situ analyzing binding kinetics between antibody and the surface protein biomarkers on the captured exosomes. Finally, the PSM can be easily constructed on a popular prism-coupled SPR system with commercially available components. Thus, it may provide an economical and powerful tool for clinical exosome analysis and exploration of fundamental issues such as exosome biomarker binding properties.

Compared with fluorescence imaging utilizing fluorophores whose lifetimes are in the order of nanoseconds, time-resolved fluorescence microscopy has more advantages in monitoring target fluorescence. In this work, compound DCF-MPYM, which is based on a fluorescein derivative, showed long-lived luminescence (22.11 μs in deaerated ethanol) and was used in time-resolved fluorescence imaging in living cells. Both nanosecond time-resolved transient difference absorption spectra and time-correlated single-photon counting (TCSPC) were employed to explain the long lifetime of the compound, which is rare in pure organic fluorophores without rare earth metals and heavy atoms. A mechanism of thermally activated delayed fluorescence (TADF) that considers the long wavelength fluorescence, large Stokes shift, and long-lived triplet state of DCF-MPYM was proposed. The energy gap (ΔEST) of DCF-MPYM between the singlet and triplet state was determined to be 28.36 meV by the decay rate of DF as a function of temperature. The ΔE(ST) was small enough to allow efficient intersystem crossing (ISC) and reverse ISC, leading to efficient TADF at room temperature. The straightforward synthesis of DCF-MPYM and wide availability of its starting materials contribute to the excellent potential of the compound to replace luminescent lanthanide complexes in future time-resolved imaging technologies.

The past decade has witnessed theoretical and experimental debates on the extraordinary long lifetime and low contact angle of surface nanobubbles. While several kinds of imaging techniques have provided promising evidence on the lifetime and gaseous nature of single surface nanobubble, each of them suffered from its own limitations before a consensus can be reached. In the present work, we employ a recently developed surface plasmon resonance microscopy (SPRM) to nonintrusively visualize single sub-100-nm surface nanobubble without labeling for the first time. The quantitative dependence between optical signal and nanobubble volume allows for resolving the dissolution kinetics, which is a key for understanding the lifetime. A superlocalization method is further introduced to monitor the trajectory of its mass center during dissolution, which uncovers the stick-slip behavior in the early stage and the migration behavior in the late stage. The label-free, nonintrusive, quantitative and sensitive features of SPRM and the potential compatibility with atomic force microscopy shed new light on the long-standing puzzle behind surface nanobubbles.

Imaging of Cell/Substrate Contacts of Living Cells with Surface Plasmon Resonance Microscopy

Unlike conventional surface plasmon resonance (SPR) using an antifouling film to anchor biomolecules and a reference channel for background subtraction, SPR microscopy for single-cell analysis uses a protein- or polypeptide-modified gold substrate to immobilize cells and a cell-free area as the reference. In this work, we show that such a substrate is prone to nonspecific adsorption (NSA) of species from the cell culture media, resulting in false background signals that cannot be correctly subtracted. To obtain accurate kinetic results, we patterned a dual-channel substrate using a microfluidic device, with one channel having poly-l-lysine deposited in situ onto a preformed polyethylene glycol (PEG) self-assembled monolayer for cell immobilization and the other channel remaining as PEG-covered for reference. The two 2.0 mm-wide channels are separated by a 75 μm barrier, and parts of the channels can be readily positioned into the field of view of an SPR microscope. The use of this dual-channel substrate for background subtraction is contrasted with the conventional approach through the following binding studies: (1) wheat germ agglutinin (WGA) attachment to the N-acetyl glucosamine and N-acetyl-neuraminic acid sites of glycans on HFF cells, and (2) the S1 protein of the COVID-19 virus conjugation with angiotensin-converting enzyme 2 (ACE2) on the HEK293 cells. Both studies revealed that interferences by NSA and the surface plasmon polariton wave diffracted by cells can be excluded with the dual-channel substrate, and the much smaller refractive index changes caused by the injected solutions can be correctly subtracted. Consequently, sensorgrams with higher signal-to-noise ratios and shapes predicted by the correct binding model can be obtained with accurate kinetic and affinity parameters that are more biologically relevant. The affinity between S1 protein and ACE2 is comparable to that measured with recombinant ACE2, yet the binding kinetics is different, suggesting that the cell membrane does impose a kinetic barrier to their interaction.

Since the first use of the surface plasmon resonance (SPR) technology for biosensing more than two decades ago, SPR has become a powerful tool for characterizing and quantifying biomolecular interactions, especially in pharmaceutical industry for drug discovery. It has made great strides both in instrumentation developments and applications. One of the most significant developments is the SPR microscopy, which integrates the optical microscopy with SPR technology to enable many new studies such as direct measurements of small drug molecules interacting with GPCR proteins on cell membrane in their native state, and direct measurements of drug response over hundreds of cells with different phenotypes and growth stages to reveal their heterogeneity and other important functionalities. Here I will present some of the latest developments in SPR technology along with its applications in diverse fields.