Measuring the binding kinetics of single proteins represents one of the most important and challenging tasks in protein analysis. Here we show that this is possible using a surface plasmon resonance (SPR) scattering technique. SPR is a popular label-free detection technology because of its extraordinary sensitivity, but it has never been used for imaging single proteins. We overcome this limitation by imaging scattering of surface plasmonic waves by proteins. This allows us to image single proteins, measure their sizes, and identify them based on their specific binding to antibodies. We further show that it is possible to quantify protein binding kinetics by counting the binding of individual molecules, providing a digital method to measure binding kinetics and analyze heterogeneity of protein behavior. We anticipate that this imaging method will become an important tool for single protein analysis, especially for low volume samples, such as single cells.

This protocol describes a method combining phase-contrast and fluorescence microscopy, Raman spectroscopy and optical tweezers to characterize the germination of single bacterial spores. The characterization consists of the following steps: (i) loading heat-activated dormant spores into a temperature-controlled microscope sample holder containing a germinant solution plus a nucleic acid stain; (ii) capturing a single spore with optical tweezers; (iii) simultaneously measuring phase-contrast images, Raman spectra and fluorescence images of the optically captured spore at 2- to 10-s intervals; and (iv) analyzing the acquired data for the loss of spore refractility, changes in spore-specific molecules (in particular, dipicolinic acid) and uptake of the nucleic acid stain. This information leads to precise correlations between various germination events, and takes 1-2 h to complete. The method can also be adapted to use multi-trap Raman spectroscopy or phase-contrast microscopy of spores adhered on a cover slip to simultaneously obtain germination parameters for multiple individual spores.

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.

Detection and identification of proteins are typically achieved by analyzing protein size, charge, mobility and binding to antibodies, which are critical for biomedical research and disease diagnosis and treatment. Despite the importance, measuring these quantities with one technology and at the single-molecule level has not been possible. Here we tether a protein to a surface with a flexible polymer, drive it into oscillation with an electric field, and image the oscillation with a near field optical imaging method, from which we determine the size, charge, and mobility of the protein. We also measure antibody binding and conformation changes in the protein. The work demonstrates a capability for comprehensive protein analysis and precision protein biomarker detection at the single molecule level.

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...

Hypoxia-responsive fluorescent probes have emerged as a novel scaffold for tumor diagnosis. However, dilemma often exists between simple synthesis and high water solubility in traditional probes. Owing to the intrinsic property of N-oxides, herein, a new strategy is proposed to design and synthesize probes for in vitro hypoxia imaging. Equipped with tetraphenylethene (TPE), the N-oxides exhibit aggregation-induced emission characteristics and emit no light in aqueous solutions. Interestingly, the N-oxides can be reduced by ferrous ions in different rates. The aggregation of the resulting hydrophobic TPE residues restricts the intramolecular motions of the molecules, which "turns-on" their fluorescence. The NO covalent bond of one molecule can be specifically cleaved by cellular reductase overexpressed under hypoxic conditions, and thus turn-on hypoxia imaging in vitro is achieved. The new strategy to design hypoxia imaging probes is extremely valuable and has great potential for application in tumor diagnosis.

Lysosomal β-N-acetylhexosaminidase (Hex) has been reported to possess unique physiological performances. Detection and visualization of Hex in lysosome will be favorable to reveal the basis of its functions. However, Hex-specific fluorescent probes are rarely reported. In this study, we reported the first lysosome-targeting Hex-lighting-up aggregation-induced emission (AIE)-active fluorescent probe (GlcNAc-TPE) with remarkably large Stokes shift and high sensitivity and selectivity. GlcNAc-TPE can selectively locate in lysosome and visualize endogenous Hex in live HCT116 cells and in live mice with high stability and good biocompatibility, providing a useful AIE probe for real-time visualization of Hex in live samples.

Cell adhesion plays a critical role in cell communication, cell migration, cell proliferation, and integration of medical implants with tissues. Focal adhesions physically link the cell cytoskeleton to the extracellular matrix, but it remains challenging to image single focal adhesions directly. Here, we show that plasmonic scattering microscopy (PSM) can directly image the single focal adhesions in a label-free, real-time, and non-invasive manner with sub-micrometer spatial resolution. PSM is developed based on surface plasmon resonance (SPR) microscopy, and the evanescent illumination makes it immune to the interference of intracellular structures. Unlike the conventional SPR microscopy, PSM can provide a high signal-to-noise ratio and sub-micrometer spatial resolution for imaging the analytes with size down to a single-molecule level, thus allowing both the super-resolution lateral localization for measuring the nanoscale displacement and precise tracking of vertical distances between the analyte centroid and the sensor surface for analysis of free-energy profiles. PSM imaging of the RBL-2H3 cell with temporal resolution down to microseconds shows that the focal adhesions have random diffusion behaviors in addition to their directional movements during the antibody-mediated activation process. The free-energy mapping also shows a similar movement tendency, indicating that the cell may change its morphology upon varying the binding conditions of adhesive structures. PSM provides insights into the individual focal adhesion activities and can also serve as a promising tool for investigating the cell/surface interactions, such as cell capture and detection and tissue adhesive materials screening.