Ying Zhao

Single‐molecule tracking (SMT) has revolutionized our understanding of molecular interactionsby revealing nanoscale behaviors and dynamic processes obscured in ensemble‐averaged measurements. While fluorescent SMT has been instrumental, its utility is constrained by fundamental limitations such as photobleaching and finite photon budgets, which restrict temporal resolution and observation periods. Furthermore, the fluorescent labels themselves can perturb native molecular interactions. The recent emergence of nonfluorescent SMT techniques offers a powerful alternative, enabling long‐term or label‐free observation with high temporal resolution for a more direct view into nanoscale binding kinetics. This review comprehensively summarizes recent advances in nonfluorescent SMT methodologies. We describe the typical optical devices, detail the tracking algorithms, and showcase their applications in elucidating previously inaccessible aspects of molecular binding. We further critically discuss persistent technical hurdles and outline promising future directions, underscoring the interdisciplinary synergy between advanced optical techniques and data science required to unlock the full potential of these next‐generation tools.

Serum albumin, a reverse acute-phase protein, tends to decrease in response to acute clinical conditions. We hypothesized that albumin levels would exhibit state-dependent dynamics, with distinct patterns between acute episodes and remission states in schizophrenia. To test this, we conducted a retrospective longitudinal study to investigate the dynamic serum albumin levels in 148 schizophrenia patients, starting from their first episode through remission and subsequent relapse. A matched general population sample served as the control group. Classification models were developed using albumin levels (Albumin _current) and changes (ΔAlbumin₁ = Albumin _current−Albumin _{previous remission}, and ΔAlbumin₂ = Albumin _current−Albumin _{previous acute episode}) to distinguish clinical states. Model performance was evaluated using the area under the receiver operating characteristic (ROC) curve (AUC). Serum albumin levels were significantly lower during acute episodes (first episode: 45·6, standard deviation (SD) 4·0 g/L; relapse: 44·9, SD 4·0 g/L) compared to remission (48·6, SD 2·9 g/L) and matched controls (48·6, SD 3·4 g/L). Patients in remission showed albumin levels comparable to controls. These findings remained consistent after adjustment for potential confounders using mixed-effects model and in sex-stratified analyses. The classification model incorporating ΔAlbumin₁ and ΔAlbumin₂ achieved an AUC of 0·88 (95% CI: 0·84, 0·92) in distinguishing acute episodes from remission. These findings highlight serum albumin’s potential as a clinically useful biomarker of illness activity/state and mental stress in schizophrenia, with utility in differentiating between acute and remitted states.

Ligand binding to membrane proteins initiates numerous therapeutic processes. Surface plasmon resonance (SPR), a popular method for analyzing molecular interactions, has emerged as a promising tool for in situ determination of membrane protein binding kinetics owing to its label-free detection, high surface sensitivity, and resistance to intracellular interference. However, the excitation of SPR relies on noble metal films, typically gold, which are biologically incompatible and can cause fluorescence quenching. Here we present a surface-sensitive waveguide imaging technique that retains the advantages of SPR and can be easily integrated into standard SPR devices by depositing additional dielectric layers onto SPR sensor chips using conventional vacuum evaporation. This novel sensor provides a silica surface, a well-known biologically compatible surface, for cell attachment and produces a sharper resonance curve and a stronger surface electric field than SPR, leading to improved measurement precision. After incorporating amplitude modulation, this approach increases measurement precision by approximately eight times compared to traditional SPR with the same optical setup. This high-precision method enables in situ single-cell analysis, revealing cell-to-cell heterogeneity that provides fuel for drug resistance. We believe this technique provides a streamlined strategy to enhance SPR for expanding their applications in biochemical research and drug screening.

Membrane proteins are crucial for various cellular processes and are key targets in pharmacological research. Their interactions with ligands are essential for elucidating cellular mechanisms and advancing drug development. To study these interactions without altering their functional properties in native environments, several advanced optical imaging methods have been developed for in situ and label-free quantification. This review focuses on recent optical imaging techniques such as surface plasmon resonance imaging (SPRi), surface plasmon resonance microscopy (SPRM), edge tracking approaches, and surface light scattering microscopy (SLSM). We explore the operational principles, recent advancements, and the scope of application of these methods. Additionally, we address the current challenges and explore the future potential of these innovative optical imaging strategies in deepening our understanding of biomolecular interactions and facilitating the discovery of new therapeutic agents.

Surface plasmon resonance (SPR) biosensors are extensively utilized for analyzing molecular interactions due to their high sensitivity and label-free detection capabilities. Recent innovations in surface-sensitive biosensors with dielectric surfaces address the inherent limitations associated with traditional gold surfaces, such as thermal effects and biocompatibility issues, which can impede broader applications. This review examines state-of-the-art biosensor configurations, including total internal reflection, optical waveguide, photonic crystal resonators, Bloch surface wave biosensors, and surface electrochemical biosensors, which can enhance analyte signals and augment the molecular detection efficiency at the sensor interface. These technological advancements not only improve the resolution of binding kinetics analysis and single-molecule detection but also extend the analytical capabilities of these systems. Additionally, this review explores prospective advancements in augmenting field enhancement and incorporating multimodal sensing functionalities, emphasizing the significant potential of these sophisticated biosensing technologies to profoundly enhance our understanding of molecular interactions.

Measuring ligand interactions with membrane proteins in single live cells is critical for understanding many cellular processes and screening drugs. However, developing such a capability has been a difficult challenge. Here, we employ evanescent scattering microscopy (ESM) to show that ligand binding to membrane proteins can change the cell adhesion properties, which are intrinsic cell properties and independent of random cell micromotions and ligand mass, thus allowing the kinetics analyses of both proteins and small molecules binding to membrane proteins in both single fixed and live cells. In addition, utilizing the high spatiotemporal resolution of ESM, the positions of cell adhesion sites can be tracked in real-time to analyze the cell deformations and migrations, thus providing a potential approach for understanding the cell activity during the ligand binding process in detail. The presented method may pave the road for developing a versatile and easy-to-use label-free detection strategy for in situ analysis of molecular interaction dynamics in living biosystems with single-cell resolution.

The emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has generated recurring worldwide infection outbreaks. These highly mutated variants reduce the effectiveness of current coronavirus disease 2019 (COVID-19) vaccines, which are designed to target only the spike (S) protein of the original virus. Except for the S of SARS-CoV-2, the immunoprotective potential of other structural proteins (nucleocapsid, N; envelope, E; membrane, M) as vaccine target antigens is still unclear and worthy of investigation. In this study, synthetic DNA vaccines encoding four SARS-CoV-2 structural proteins (pS, pN, pE, and pM) were developed, and mice were immunized with three doses via intramuscular injection and electroporation. Notably, co-immunization with two DNA vaccines that expressed the S and N proteins induced higher neutralizing antibodies and was more effective in reducing the SARS-CoV-2 viral load than the S protein alone in mice. In addition, pS co-immunization with either pN or pE + pM induced a higher S protein-specific cellular immunity after three immunizations and caused milder histopathological changes than pS alone post-challenge. The role of the conserved structural proteins of SARS-CoV-2, including the N/E/M proteins, should be investigated further for their applications in vaccine design, such as mRNA vaccines.