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The advancement of optical microscopy technologies has achieved imaging of nanoscale objects, including nanomaterials, virions, organelles, and biological molecules, at the single entity level. Recently developed plasmonic and scattering based optical microscopy technologies have enabled label-free imaging of single entities with high spatial and temporal resolutions. These label-free methods eliminate the complexity of sample labeling and minimize the perturbation of the analyte native state. Additionally, these imaging-based methods can noninvasively probe the dynamics and functions of single entities with sufficient throughput for heterogeneity analysis. This perspective will review label-free single entity imaging technologies and discuss their principles, applications, and key challenges.

Most label-free techniques rely on measuring refractive index or mass change on the sensor surface. Thus, it is challenging for them to measure small molecules or enzymatic processes that only induce a minor mass change on the analyte molecules. Here, we have developed a technique by combining Surface Plasmon Resonance sensing with an Oscillating Biomolecule Layer approach (SPR-OBL) to enhance the sensitivity of traditional SPR. In addition to the inherent mass sensitivity, SPR-OBL is also sensitive to the charge and conformational change of the analyte; hence it overcomes the mass limit and is able to detect small molecules. We show that the multimetric SPR-OBL measurement allows for sensing any changes regarding mass, charge, and conformation, which expands the detection capability of SPR.

Molecular interactions in live cells play an important role in both cellular functions and drug discovery. Current methods for measuring binding kinetics involve extracting the membrane protein and labeling, while the in situ quantification of molecular interaction with surface plasmon resonance (SPR) imaging mainly worked with fixed cells due to the micro-motion related noises of live cells. Here, an optical imaging method is presented to measure the molecular interaction with live red blood cells by tracking the nanometer membrane fluctuations. The membrane fluctuation dynamics are measured by tracking the membrane displacement during glycoprotein interaction. The data are analyzed with a thermodynamic model to determine the elastic properties of the cell observing reduced membrane fluctuations under fixatives, indicating cell fixations affect membrane mechanical properties. The binding kinetics of glycoprotein to several lectins are obtained by tracking the membrane fluctuation amplitude changes on single live cells. The binding kinetics and strength of different lectins are quite different, indicating the glycoproteins expression heterogeneity in single cells. It is anticipated that the method will contribute to the understanding of mechanisms of cell interaction and communication, and have potential applications in the mechanical assessment of cancer or other diseases at the single-cell level, and screening of membrane protein targeting drugs., (© 2022 Wiley-VCH GmbH.)

Quantification of molecular interactions on a surface is typically achieved via label-free techniques such as surface plasmon resonance (SPR). The sensitivity of SPR originates from the characteristic that the SPR angle is sensitive to the surface refractive index change. Analogously, in another interfacial optical phenomenon, total internal reflection, the critical angle is also refractive index dependent. Therefore, surface refractive index change can also be quantified by measuring the reflectivity near the critical angle. Based on this concept, we develop a method called critical angle reflection (CAR) imaging to quantify molecular interactions on glass surface. CAR imaging can be performed on SPR imaging setups. Through a side-by-side comparison, we show that CAR is capable of most molecular interaction measurements that SPR performs, including proteins, nucleic acids and cell-based detections. In addition, we show that CAR can detect small molecule bindings and intracellular signals beyond SPR sensing range. CAR exhibits several distinct characteristics, including tunable sensitivity and dynamic range, deeper vertical sensing range, fluorescence compatibility, broader wavelength and polarization of light selection, and glass surface chemistry. We anticipate CAR can expand SPR's capability in small molecule detection, whole cell-based detection, simultaneous fluorescence imaging, and broader conjugation chemistry.

Measuring molecular binding is critical for understanding molecular-scale biological processes and screening drugs. Label-free detection technologies, such as surface plasmon resonance (SPR), have been developed for analyzing analytes in their natural forms. However, the specificity of these methods is solely relying on surface chemistry and has often nonspecific binding issues when working with samples in complex media. Herein, we show that single-molecule-based measurement can distinct specific and nonspecific binding processes by quantifying the mass and binding dynamics of individual-bound analyte molecules, thus allowing the binding kinetic analysis in complex media such as serum. In addition, this single-molecule imaging is realized in a commonly used Kretschmann prism-coupled SPR system, thus providing a convenient solution to realize high-resolution imaging on widely used prism-coupled SPR systems.

Surface plasmon resonance microscopy (SPRM) is a versatile platform for chemical and biological sensing and imaging. Great progress in exploring its applications, ranging from single-molecule sensing to single-cell imaging, has been made. In this Minireview, we introduce the principles and instrumentation of SPRM. We also summarize the broad and exciting applications of SPRM to the analysis of single entities. Finally, we discuss the challenges and limitations associated with SPRM and potential solutions., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report on imaging of local electric field on an electrode surface with plasmonic electrochemical impedance microscopy (P-EIM). The local electric field is created by putting an electrode inside a micropipet positioned over the electrode and applying a voltage between the two electrodes. We show that the distribution of the surface charge as well as the local electric field at the electrode surface can be imaged with P-EIM. The spatial distribution and the dependence of the local charge density and electric field on the distance between the micropipet and the surface are measured, and the results are compared with the finite element calculations. The work also demonstrates the possibility of integrating plasmonic imaging with scanning ion conductance microscopy (SICM) and other scanning probe microscopies.

Epidermal growth factor receptor (EGFR, also known as ErbB-1 or HER-1) is a membrane bound protein that has been associated with a variety of solid tumors and the control of cell survival, proliferation, and metabolism. Quantification of the EGFR expression level in cell membranes and the interaction kinetics with drugs are thus important for cancer diagnosis and treatment. Here we report mapping of the distribution and interaction kinetics of EGFR in their native environment with the surface plasmon resonance imaging (SPRi) technique. The monoclonal anti-EGFR antibody was used as a model drug in this study. The binding of the antibody to EGFR overexpressed A431 cells was monitored in real time, which was found to follow the first-order kinetics with an association rate constant (ka) and dissociation rate constant (kd) of (2.7 ± 0.6) × 10(5) M(-1) s(-1) and (1.4 ± 0.5) × 10(-4) s(-1), respectively. The dissociation constant (KD) was determined to be 0.53 ± 0.26 nM with up to seven-fold variation among different individual A431 cells. In addition, the averaged A431 cell surface EGFR density was found to be 636/μm(2) with an estimation of 5 × 10(5) EGFR per cell. Additional measurement also revealed that different EGFR positive cell lines (A431, HeLa, and A549) show receptor density dependent anti-EGFR binding kinetics. The results demonstrate that SPRi is a valuable tool for direct quantification of membrane protein expression level and ligand binding kinetics at single cell resolution. Our findings show that the local environment affects the drug-receptor interactions, and in situ measurement of membrane protein binding kinetics is important.

We report on a plasmonics-based electrochemical current imaging of redox reactions in aqueous droplets with diameters varying from a few hundred nanometers (tens of attoliter in volume) to a few micrometers. The imaging technique allows us to obtain cyclic voltammograms of multiple droplets on a gold electrode simultaneously and to examine the local redox reactions within a droplet. The results are supported by numerical simulations. The work demonstrates a new capability of studying electrochemistry in microdroplets, which offers an opportunity to understand electrochemical reactions within a small confined volume.

Surface plasmon resonance (SPR) has become an indispensable tool for label-free detection and quantification of molecular binding. Traditionally, the principle of SPR biosensors is described with a stratified medium model, in which discrete molecules are approximated with a uniform thin film. With the recent technical advances, SPR can now detect extremely low coverage of molecules, which raises the question of the validity of the traditional model. Here, we present combined theoretical, numerical and experimental analysis of SPR detection principle by considering the discrete nature of the molecules (particles). Our results show that the stratified medium model can provide reasonable description of SPR biosensors for relatively high coverage and weakly scattering samples. However, interference between the SPR images of individual particles needs to be considered for high spatial resolution images and for strong scattering samples at certain incident angles of light.

Detection of a single or small amount of charges and molecules in biologically relevant aqueous solutions is a long-standing goal in analytical science and detection technology. Here we report on self-assembled nano-oscillators for charge and molecular binding detections in aqueous solutions. Each nano-oscillator consists of a nanoparticle linked to a solid surface via a molecular tether. By applying an oscillating electric field normal to the surface, the nanoparticles oscillate, which is detected individually with ∼0.1 nm accuracy by a plasmonic imaging technique. From the oscillation amplitude and phase, the charge of the nanoparticles is determined with a detection limit of ∼0.18 electron charges along with the charge polarity. We further demonstrate the detection of molecular binding with the self-assembled nano-oscillators.

The capability of imaging and detecting single DNA molecules is critical in the study, analysis, and applications of DNA. Fluorescence imaging is a widely used method, but it suffers from blinking and photobleaching, and fluorescence tags may block or affect binding sites on DNA. We report on label-free imaging of single DNA molecules with a differential plasmonic imaging technique. The technique produces high contrast images due to the scattering of surface plasmonic waves by the molecules and the removal of background noises and interference patterns, allowing for quantitative analysis of individual DNA molecules. Simulation of the images based on a scattering model shows good agreement with the experiment. We further demonstrate optical mapping of single DNA molecules.

A simple device for high-speed microfluidic delivery of liquid samples to a surface plasmon resonance sensor surface is presented. The delivery platform is comprised of a four-port microfluidic cell, two ports serve as inlets for buffer and sample solutions, respectively, and a high-speed selector valve to control the alternate opening and closing of the two outlet ports. The time scale of buffer/sample switching (or sample injection rise and fall time) is on the order of milliseconds, thereby minimizing the opportunity for sample plug dispersion. The high rates of mass transport to and from the central microfluidic sensing region allow for SPR-based kinetic analysis of binding events with dissociation rate constants (k(d)) up to 130 s(-1). The required sample volume is only 1 μL, allowing for minimal sample consumption during high-speed kinetic binding measurement.

Charge-based detection of small molecules is demonstrated by plasmonic-based electrochemical impedance microscopy (P-EIM). The dependence of surface plasmon resonance (SPR) on surface charge density is used to detect small molecules (60-120 Da) printed on a dextran-modified sensor surface. Local variations in charge density on an electrode surface are manifest in an optical SPR signal. The SPR response to an applied ac potential measures the sensor surface impedance which is a function of the surface charge density. This optical signal is comprised of a dc and an ac component, and is measured with high spatial resolution. The dc element of the SPR signal represents conventional SPR imaging information. The amplitude and phase of local surface impedance is provided by the ac component. The phase signal of the small molecules is a function of their charge status, which is manipulated by the pH of a solution. Small molecules with positive, neutral, and negative charge are detected by P-EIM. This technique is used to detect and distinguish small molecules based on their charge status, thereby circumventing the mass limitation (~100 Da) of conventional SPR measurement.

We report the imaging of the cell-substrate adhesion of a single cell with subcellular spatial resolution. Osmotic pressure was introduced to provide a controllable mechanical stimulation to the cell attached to a substrate, and high-resolution surface plasmon resonance microscopy was used to map the response of the cell, from which local cell-substrate adhesion was determined. In addition to high spatial resolution, the approach is noninvasive and fast and allows for the continuous mapping of cell-substrate interactions and single-cell movements.

Plasmonic-based electrochemical impedance spectroscopy (P-EIS) is developed to investigate molecular binding on surfaces. Its basic principle relies on the sensitive dependence of surface plasmon resonance (SPR) signal on surface charge density, which is modulated by applying an ac potential to a SPR chip surface. The ac component of the SPR response gives the electrochemical impedance, and the dc component provides the conventional SPR detection. The plasmonic-based impedance measured over a range of frequency is in quantitative agreement with the conventional electrochemical impedance. Compared to the conventional SPR detection, P-EIS is sensitive to molecular binding taking place on the chip surface and less sensitive to bulk refractive index changes or nonspecific binding. Moreover, this new approach allows for simultaneous SPR and surface impedance analysis of molecular binding processes., (© 2011 American Chemical Society)

The serine protease coagulation factor thrombin functions primarily in hemostasis, but is also involved in atherosclerosis, thromboembolic disease, cancer and inflammatory disease. Direct measurement of coagulation proteins including thrombin in plasma samples poses a significant challenge because of lack of specific probes and low thrombin concentrations. In addition, high plasma protein concentrations in samples can result in high backgrounds. These challenges were overcome using a bi-cell surface plasmon resonance (SPR) spectrometer with an immobilized thrombin aptamer to measure thrombin in samples passed through a low volume flow cell. For thrombin in Tris-EDTA buffer, the limit of detection (LOD) was 25 nM. Coefficient of variation (CV) for detection of 50 nM was 12.2% and 12.4% for intra and inter-day measurements respectively. This detection was specific for both thrombin aptamer and for thrombin. Using serum samples spiked with thrombin, the LOD was 50 nM with a linear range of detection from 50 nM to 200 nM. However use of serum samples was associated with consistent, low-level background drift. The contributions of nonspecific protein absorption onto the sensor surface and sample flow speed were assessed, and strategies to reduce this background drift were explored. We conclude that the bi-cell SPR platform with an aptamer capture probe can be employed as a highly sensitive real-time, label-free biosensor for the detection of coagulation factors in plasma samples., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Electrochemical impedance spectroscopy is a crucial tool for the detection and study of various biological substances, from DNA and proteins to viruses and bacteria. It does not require any labelling species, and methods based on it have been developed to study cellular processes (such as cell spreading, adhesion, invasion, toxicology and mobility). However, data have so far lacked spatial information, which is essential for investigating heterogeneous processes and imaging high-throughput microarrays. Here, we report an electrochemical impedance microscope based on surface plasmon resonance that resolves local impedance with submicrometre spatial resolution. We have used an electrochemical impedance microscope to monitor the dynamics of cellular processes (apoptosis and electroporation of individual cells) with millisecond time resolution. The high spatial and temporal resolution makes it possible to study individual cells, but also resolve subcellular structures and processes without labels, and with excellent detection sensitivity (~2 pS). We also describe a model that simulates cellular and electrochemical impedance microscope images based on local dielectric constant and conductivity.

We report on label-free imaging, detection, and mass/size measurement of single viral particles in solution by high-resolution surface plasmon resonance microscopy. Diffraction of propagating plasmon waves along a metal surface by the viral particles creates images of the individual particles, which allow us to detect the binding of the viral particles to surfaces functionalized with and without antibodies. We show that the intensity of the particle image is related to the mass of the particle, from which we determine the mass and mass distribution of influenza viral particles with a mass detection limit of approximately 1 ag (or 0.2 fg/mm(2)). This work demonstrates a multiplexed method to measure the masses of individual viral particles and to study the binding activity of the viral particles.

We demonstrated an electrochemical microscopy technique based on the detection of variations in local electrochemical current from optical signals arising from surface plasmon resonance. It enables local electrochemical measurements (such as voltammetry and amperometry) with high spatial resolution and sensitivity, because the signal varies with current density rather than current. The imaging technique is noninvasive, scanning-free, and fast, and it constitutes a powerful tool for studying heterogeneous surface reactions and for analyzing trace chemicals.

A quantitative formalism of electrochemical surface plasmon resonance (EC-SPR) was developed for studying electrochemical reactions. The EC-SPR signal from the reactions was found to be a convolution function of electrochemical current, and therefore, EC-SPR is a powerful tool that can provide information similar to the conventional current-based electrochemical techniques. As an example, potential-sweep EC-SPR was analyzed in details and was found to provide a new way to measure convolution voltammetry without the need of numerical integration. In addition to the benefits provided by the conventional convolution voltammetry, the EC-SPR has several unique advantages, including (1) spatial resolution that is particularly attractive for studying heterogeneous reactions, (2) optical properties of the reactions species that may assist identification of reaction mechanisms, and (3) high surface sensitivity for studying surface binding of the reaction species. Experiments and numerical simulations were carried out for a model system, hexaammineruthenium(III) chloride. The simultaneously measured electrochemical current and SPR response confirmed the relationship between the two quantities, and the numerical simulations were in excellent agreement with the measurements.

We demonstrate a method to measure surface charge density and particle height using surface plasmon resonance (SPR) detection. It is based on two facts: (1) The equilibrium height of a charged particle over a charged surface depends on the electrostatic interaction between the particle and the surface; and (2) SPR is extremely sensitive to the height of the particle. We perform numerical simulations to establish the relations between the SPR signal and the particle height, and between the particle height and the surface charge density, and carry out systematic experiments, including effects of different buffer concentrations, particle sizes, and concentrations, to examine the relations. The simulation and experimental results are in good agreement with each other. Using the method, we determine surface charge density of gold surface functionalized with different molecules. If the surface charge density is known, the method can also be used to determine the charges of the particles.

High-resolution differential surface plasmon resonance (SPR) with anodic stripping voltammetry (ASV) capability has been demonstrated for detecting heavy metal ions in water. Metal ions are electroplated onto the gold SPR sensing surface and are quantitatively detected by stripping voltammetry. Both the SPR angular shift and electrochemical current signal are recorded to identify the type and amount of the metal ions in water. The performance of the combined approach is further enhanced by a differential detection approach. The gold sensor surface is divided into a reference and a sensing area, and the difference in the SPR angles from the two areas is detected with a quadrant cell photodetector as a differential signal. Our system demonstrated quantitative detection of copper, lead, and mercury ions in water from part-per-million to sub-part-per-billion levels with good linearity.

Measuring molecular binding kinetics represents one of the most important tasks in molecular interaction analysis. Surface plasmon resonance (SPR) is a popular tool for analyzing molecular binding. Plasmonic scattering microscopy (PSM) is a newly developed SPR imaging technology, which detects the out‐of‐plane scattering of surface plasmons by analytes and has pushed the detection limit of label‐free SPR imaging down to a single‐protein level. In addition, PSM also allows SPR imaging with high spatiotemporal resolution, making it possible to analyze cellular response to the molecular bindings. In this Mini Review, we present PSM as a method of choice for chemical and biological imaging, introduce its theoretical mechanism, present its experimental schemes, summarize its exciting applications, and discuss its challenges as well as the promising future. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Surface plasmon resonance microscopy (SPRM) is a versatile platform for chemical and biological sensing and imaging. Great progress in exploring its applications, ranging from single‐molecule sensing to single‐cell imaging, has been made. In this Minireview, we introduce the principles and instrumentation of SPRM. We also summarize the broad and exciting applications of SPRM to the analysis of single entities. Finally, we discuss the challenges and limitations associated with SPRM and potential solutions. [ABSTRACT FROM AUTHOR]

Abstract: The electrochemical reaction of hydroquinone–benzoquinone (HQ–BQ) system in acetonitrile was studied by a flow-through Electrochemical Surface Plasmon Resonance (EC-SPR) technique. The semiquinone radical anion (BQ•−) was detected as a large negative SPR shift for the first time by the hyphenated technique. The real time SPR signal shows the entire reaction process of the BQ•− including generation, transformation and disappearance. The lifetime of the BQ•− in the reaction was extracted by comparing the SPR signals along the flow stream. The study demonstrates that the flow-through EC-SPR is a powerful tool for monitoring not only surface bindings, but also solution phase chemical reactions. In addition, it is especially suitable for detecting unstable intermediate reaction products, which are difficult to measure using conventional analytical methods. [ABSTRACT FROM AUTHOR]

We demonstrated a method to accurately measure the zeta potentials and surface charges of individual particles by surface plasmon resonance microscopy (SPRM). The principle is based on the sensitive dependence of surface plasmons in a metal surface on the distance between a particle and the surface. By applying a periodic (ac) electric field to the metal surface, the charged particle oscillates, which is measured with SPRM, from which the zeta potential and the surface charge of the particle can be determined. The ac method reduces the electro-osmotic effect and noises induced by Brownian motions and allows for the rapid determination of the zeta potentials of individual particles. [ABSTRACT FROM AUTHOR]

Most drugs work by binding to receptors on the cell surface. Quantification of binding kinetics between drug and membrane protein is an essential step in drug discovery. Current methods for measuring binding kinetics involve extracting the membrane protein and labeling, and both have issues. Surface plasmon resonance (SPR) imaging has been demonstrated for quantification of protein binding to cells with single-cell resolution, but it only senses the bottom of the cell and the signal diminishes with the molecule size. We have discovered that ligand binding to the cell surface is accompanied by a small cell membrane deformation, which can be used to measure the binding kinetics by tracking the cell edge deformation. Here, we report the first integration of SPR imaging and cell edge tracking methods in a single device, and we use lectin interaction as a model system to demonstrate the capability of the device. The integration enables the simultaneous collection of complementary information provided by both methods. Edge tracking provides the advantage of small molecule binding detection capability, while the SPR signal scales with the ligand mass and can quantify membrane protein density. The kinetic constants from the two methods were cross-validated and found to be in agreement at the single-cell level. The variation of observed rate constant between the two methods is about 0.009 s−1, which is about the same level as the cell-to-cell variations. This result confirms that both methods can be used to measure whole-cell binding kinetics, and the integration improves the reliability and capability of the measurement. [ABSTRACT FROM AUTHOR]

TiO 2 was successfully synthesized within the internal pores of MIL-101(Cr), leading to the formation of a TiO 2 -in-MIL-101(Cr) composite. This heterojunction structure significantly improved the photogenerated electron-hole separation within the metal-organic framework (MOF) composite. Subsequently, carbon dots (CDs) and silver nanoparticles (AgNPs) were introduced to further modify the TiO 2 -in-MIL-101(Cr), resulting in a multimodified MIL-101(Cr) composite. The integration of CDs enhanced the light absorption capabilities of the composite, thereby boosting the photocurrent. Meanwhile, AgNPs not only enhanced the absorption of visible light through the local surface plasmon resonance effect (LSPR) but also facilitated efficient electron transport. This multimodal modification strategy led to a remarkable enhancement in the photoelectrochemical (PEC) performance of MIL-101(Cr), with the photocurrent density (J) increasing by approximately a factor of 19. To further expand the functionality of this composite, AβO capture DNA (cDNA) and CuS-binding aptamer were grafted onto the TiO 2 -in-MIL-101(Cr)@CDs@AgNPs, creating a sandwich-like structure. Based on this multimodified MIL-101(Cr) composite, an "on-off-on" type PEC biosensor was constructed. The p -type semiconductor CuS served as a signal amplifier, capturing photogenerated electrons and effectively reducing the photocurrent. When cDNA hybridized with AβO, the Apt-CuS moiety detached from the MIL-101(Cr) composite, leading to the restoration of the photocurrent. The PEC biosensor for the detection of AβO exhibited optimal performance at a pH of 7.00, an incubation temperature of 37 °C, and an incubation time of 45 min. Under these conditions, the biosensor demonstrated a wide detection range of 5 fM to 1 μM, with an ultralow detection limit of 4.36 fM. This method exhibits high sensitivity, robust anti-interference capabilities, and holds great promise for applications in tracing the detection of AβO in human serum. [Display omitted] • TiO 2 -in-MIL-101(Cr) enhanced electron-hole separation through heterojunction structure. • CDs and AgNPs modification significantly improved PEC performance of MIL-101(Cr). • CuS as signal amplifier effectively improved the sensitivity of the PEC biosensor. • AβO detection PEC biosensor based on TMCA exhibited high sensitivity and low LOD. • The "on-off-on" PEC method enabled precise AβO tracing in human serum. [ABSTRACT FROM AUTHOR]

Fluorescent labeling is a mainstream technology for detecting molecular binding. Despite the importance, few studies have been devoted to quantitatively examine the effect of labeling on the molecular binding processes. Here we present a quantitative study on the binding kinetics of fluorescent-labeled and un-labeled molecules (lectin proteins) with glycoproteins on the membrane of cells using surface plasmon resonance imaging (SPRi) technique. The study shows that fluorescent labeling has a significant influence on the binding behaviors of proteins, especially the association processes, and the influence depends sensitively on the charge of fluorescent labels. It further shows that the labels also affect the local distribution of probe proteins, due to the inhomogeneous surface charge distribution of the cell membrane. Our work indicates that fluorescent labeling in general affects the binding behaviors, but proper design of the label will help to minimize its effect. [ABSTRACT FROM AUTHOR]

Quantifying the interactions of bacteria with external ligands is fundamental to the understanding of pathogenesis, antibiotic resistance, immune evasion, and mechanism of antimicrobial action. Due to inherent cell-to-cell heterogeneity in a microbial population, each bacterium interacts differently with its environment. This large variability is washed out in bulk assays, and there is a need of techniques that can quantify interactions of bacteria with ligands at the single bacterium level. In this work, we present a label-free and real-time plasmonic imaging technique to measure the binding kinetics of ligand interactions with single bacteria, and perform statistical analysis of the heterogeneity. Using the technique, we have studied interactions of antibodies with single Escherichia coli O157:H7 cells and demonstrated a capability of determining the binding kinetic constants of single live bacteria with ligands, and quantify heterogeneity in a microbial population. [ABSTRACT FROM AUTHOR]