Single Multifunctional Nanocabinets-Based Target-Activated Feedback for Simultaneously Precise Monitoring and Therapy.

Stimulus-responsive mode is highly desirable for improving the precise monitoring and physiological efficacy of endogenous biomarkers (EB). However, its integrated application for visual detection and therapy is limited by inappropriate use of responsive triggers and poor delivery of EB signal-transducing agents, which remain challenging in simultaneous monitoring and noninvasive therapy of EB and EB-mediated pathological events. Target microRNA (miRNA) as controllable reaction triggers and DNAzyme as signal-transducing agent are proposed to develop target-stimulated multifunctional nanocabinets (MFNCs) for the visual tracking of both miRNA and miRNA-mediated anticancer events. The MFNCs, equipped with a target-discriminating sequence-incorporated DNAzyme motif, can specifically release therapeutic molecules through target-triggered conformational switches, accompanied by transduction signal output. Target detection and molecule release performance are recorded in parallel via reverse dual-signal feedback at the single-molecule level. In addition, the intrinsic thermal-replenishing of the MFNCs leads to tumor ablation without invasive exogenous aids. The system achieves visual target quantification, anticancer molecule real-time tracking, and tumor suppression in vivo and in vitro. This work proposes a new paradigm for precise visual exploration of EB or EB-mediated bio-events and provides a demonstration of efficacious all-in-one detection and therapy based on the target-triggered multifunctional nanosystem.

Dual-Signal Imaging Mode Based on Fluorescence and Electrochemiluminescence for Ultrasensitive Visualization of SARS-CoV-2 Spike Protein.

Accurate and reliable detection of SARS-CoV-2 is critical for the effective prevention and rapid containment of COVID-19. Current approaches suffer from complex procedures or a single signal readout, resulting in an increased risk of false negatives and low sensitivity. Here, we developed a fluorescence (FL) and electrochemiluminescence (ECL) dual-mode imaging platform based on a self-powered DNAzyme walker to achieve accurate surveillance of SARS-CoV-2 spike protein at the single-molecule level. The specific activation of the DNAzyme walker by the target protein provides the power for the system's continuous running, enabling the simultaneous recording of the reduction in fluorescence spots and the appearance of ECL spots generated by the Ru-doped metal-organic framework (MOF) emitter. Therefore, the constructed imaging platform can achieve dual-mode detection of spike protein via reverse dual-signal feedback, which could effectively eliminate false-positive or false-negative signals and improve the detection accuracy and sensitivity with a low detection limit. In particular, the dual-mode accuracy of spike protein diagnosis in samples has been significantly improved compared to single-signal output means. In addition, this dual-mode imaging platform may become a prospective diagnostic device for other infectious viruses.

Pyroptosis-related gene signature: A predictor for overall survival, immunotherapy response, and chemosensitivity in patients with pancreatic adenocarcinoma.

Background: Pancreatic adenocarcinoma (PAAD) is a lethal malignancy with high levels of heterogeneity. Pyroptosis is thought to influence the development of various tumors. Nevertheless, the role of pyroptosis-related genes (PRGs) in prognostic risk stratification and therapeutic guidance for PAAD remains ambiguously. Methods: Transcriptome profile and clinical information of PAAD patients were retrieved from The Cancer Genome Atlas (TCGA) as well as Gene Expression Omnibus (GEO) databases, followed by differential analysis. Patients were divided into distinct pyroptosis phenotype subtypes based on the characteristic of differently expressed PRGs (DEPRGs). Then a PRG signature was established through univariate analysis and LASSO algorithm in the training set to assess the prognostic risk, and its reliability was verified in the validation set using receiver operating characteristic(ROC) curve. The correlation of risk score with tumor microenvironment(TME), TMB and chemotherapeutic drug sensitivity were also analyzed. In addition, a nomogram was constructed to promote better clinical application. Results: A total of 28 DEPRGs were determined in the integrated TCGA-GEO datasets. Patients were divided into three pyroptosis phenotype subtypes, Kaplan-Meier curve suggested patients in cluster B had a worse prognosis than those in cluster A and C. Then a price signature comprised of 8 PRGs was generated. TME analysis suggested that the low-risk subgroup displayed potential stronger antitumor immune effect and might respond better to immune checkpoint inhibitors (ICIs) therapy. Furthermore, PRG signature exhibited favorable discriminatory ability for TMB status and the sensitivity of multiple conventional chemotherapeutic agents including paclitaxel. Ultimately, we constructed a promising nomogram according to the risk score and N stage with good predictive accuracy compared with the actual overall survival (OS) probabilities. Conclusion: We established an 8-gene signature that could be regarded as an independent prognostic risk factor for PAAD patients. The 8-gene signature could provide rationale for immunotherapy and chemotherapy, which might help clinicians make precise individualized treatment regimens.

Secondary Infection Surveillance with Metagenomic Next-Generation Sequencing in COVID-19 Patients: A Cross-Sectional Study.

Background: Metagenomic next-generation sequencing (mNGS) is a promising tool for improving antimicrobial therapy and infection control decision-making in complex infections. Secondary infection surveillance using mNGS in COVID-19 patients has rarely been reported. Methods: Respiratory pathogen and antibiotic resistance prediction were evaluated by BALF mNGS for 192 hospitalized COVID-19 patients between December 2022 and February 2023. Results: Secondary infection was confirmed in 83.3% (160/192) of the COVID-19 patients, with bacterial infections (45%, 72/160) predominating, followed by mixed bacterial and fungal infections (20%, 32/160), and fungal infections (17.5%, 28/160). The incidence of bacterial or viral secondary infection was significantly higher in patients who were admitted to the ICU, received mechanical ventilation, or developed severe pneumonia (all p<0.05). Klebsiella pneumoniae (n=30, 8.4%) was the most prevalent pathogen associated with secondary infection followed by Acinetobacter baumannii (n=29, 8.1%), Candida albicans (n=29, 8.1%), Aspergillus fumigatus (n=27, 7.6%), human herpes simplex virus type 1 (n=23, 6.4%), Staphylococcus aureus (n=20, 5.6%) and Pneumocystis jiroveci (n=14, 3.9%). The overall concordance between the resistance genes detected by mNGS and the reported phenotypic resistance in 69 samples containing five clinically important pathogens (ie, K. pneumoniae, A. baumannii, S. aureus, P. aeruginosa and E. coli) that caused secondary infection was 85.5% (59/69). Conclusion: mNGS can detect pathogens causing secondary infection and predict antimicrobial resistance for COVID19 patients. This is crucial for initiating targeted treatment and rapidly detect unsuspected spread of multidrug-resistant pathogens.

Nanoconfinement-Enhanced Electrochemiluminescence for in Situ Imaging of Single Biomolecules.

Direct imaging of electrochemical reactions at the single-molecule level is of potential interest in materials, diagnostic, and catalysis applications. Electrochemiluminescence (ECL) offers the opportunity to convert redox events into photons. However, it is challenging to capture single photons emitted from a single-molecule ECL reaction at a specific location, thus limiting high-quality imaging applications. We developed the nanoreactors based on Ru(bpy)32+-doped nanoporous zeolite nanoparticles (Ru@zeolite) for direct visualization of nanoconfinement-enhanced ECL reactions. Each nanoreactor not only acts as a matrix to host Ru(bpy)32+ molecules but also provides a nanoconfined environment for the collision reactions of Ru(bpy)32+ and co-reactant radicals to realize efficient in situ ECL reactions. The nanoscale confinement resulted in enhanced ECL. Using such nanoreactors as ECL probes, a dual-signal sensing protocol for visual tracking of a single biomolecule was performed. High-resolution imaging of single membrane proteins on heterogeneous cells was effectively addressed with near-zero backgrounds. This could provide a more sensitive tool for imaging individual biomolecules and significantly advance ECL imaging in biological applications.

Direct Visualization of Nanoconfinement Effect on Nanoreactor via Electrochemiluminescence Microscopy.

Nanoconfinement in mesoporous nanoarchitectures could dramatically change molecular transport and reaction kinetics during electrochemical process. A molecular-level understanding of nanoconfinement and mass transport is critical for the applications, but a proper route to study it is lacking. Herein, we develop a single nanoreactor electrochemiluminescence (SNECL) microscopy based on Ru(bpy)3 2+ -loaded mesoporous silica nanoparticle to directly visualize in situ nanoconfinement-enhanced electrochemical reactions at the single molecule level. Meanwhile, mass transport capability of single nanoreactor, reflected as long decay time and recovery ability, is monitored and simulated with a high spatial resolution. The nanoconfinement effects in our system also enable imaging single proteins on cellular membrane. Our SNECL approach may pave the way to decipher the nanoconfinement effects during electrochemical process, and build bridges between mesoporous nanoarchitectures and potential electrochemical applications.

Single-Particle Optical Imaging for Ultrasensitive Bioanalysis.

The quantitative detection of critical biomolecules and in particular low-abundance biomarkers in biofluids is crucial for early-stage diagnosis and management but remains a challenge largely owing to the insufficient sensitivity of existing ensemble-sensing methods. The single-particle imaging technique has emerged as an important tool to analyze ultralow-abundance biomolecules by engineering and exploiting the distinct physical and chemical property of individual luminescent particles. In this review, we focus and survey the latest advances in single-particle optical imaging (OSPI) for ultrasensitive bioanalysis pertaining to basic biological studies and clinical applications. We first introduce state-of-the-art OSPI techniques, including fluorescence, surface-enhanced Raman scattering, electrochemiluminescence, and dark-field scattering, with emphasis on the contributions of various metal and nonmetal nano-labels to the improvement of the signal-to-noise ratio. During the discussion of individual techniques, we also highlight their applications in spatial-temporal measurement of key biomarkers such as proteins, nucleic acids and extracellular vesicles with single-entity sensitivity. To that end, we discuss the current challenges and prospective trends of single-particle optical-imaging-based bioanalysis.

Spatially resolved single-molecule profiling of microRNAs in migrating cells driven by microconfinement.

Cancer cells utilize a range of migration modes to navigate through a confined tissue microenvironment in vivo, while regulatory roles of key microRNAs (miRNAs) remain unclear. Precisely engineered microconfinement and the high spatial-resolution imaging strategy offer a promising avenue for deciphering the molecular mechanisms that drive cell migration. Here, enzyme-free signal-amplification nanoprobes as an effective tool are developed for three-dimensional (3D) high-resolution profiling of key miRNA molecules in single migrating cells, where distinct migration modes are precisely driven by microconfinement-engineered microchips. The constructed nanoprobes exhibit intuitive and ultrasensitive miRNA characterization in vitro by virtue of a single-molecule imaging microscope, and the differential expression and intracellular locations in different cell lines are successfully monitored. Furthermore, 3D spatial distribution of miR-141 at high resolution in flexible phenotypes of migrating cells is reconstructed in the engineered biomimetic microenvironment. The results indicate that miR-141 may be involved in the metastatic transition from a slow to a fast migration state. This work offers a new opportunity for investigating regulatory mechanisms of intracellular key biomolecules during cell migration in biomimetic microenvironments, which may advance in-depth understanding of cancer metastasis in vivo.

High Electrochemiluminescence from Ru(bpy)3 2+ Embedded Metal-Organic Frameworks to Visualize Single Molecule Movement at the Cellular Membrane.

Direct imaging of single-molecule and its movement is of fundamental importance in biology, but challenging. Herein, aided by the nanoconfinement effect and resultant high reaction activity within metal-organic frameworks (MOFs), the designed Ru(bpy)3 2+ embedded MOF complex (RuMOFs) exhibits bright electrochemiluminescence (ECL) emission permitting high-quality imaging of ECL events at single molecule level. By labeling individual proteins of living cells with single RuMOFs, the distribution of membrane tyrosine-protein-kinase-like7 (PTK7) proteins at low-expressing cells is imaged via ECL. More importantly, the efficient capture of ECL photons generated inside the MOFs results in a stable ECL emission up to 1 h, allowing the in operando visualization of protein movements at the cellular membrane. As compared with the fluorescence observation, near-zero ECL background surrounding the target protein with the ECL emitter gives a better contrast for the dynamic imaging of discrete protein movement. This achievement of single molecule ECL dynamic imaging using RuMOFs will provide a more effective nanoemitter to observe the distribution and motion of individual proteins at living cells.

Hemin-graphene oxide-gold nanoflower-assisted enhanced electrochemiluminescence immunosensor for determination of prostate-specific antigen.

An ultrasensitive luminol electrochemiluminescence (ECL) immunosensor was constructed for the detection of prostate specific antigen (PSA) using glucose oxidase-decorated hemin-graphene oxide-gold nanoflowers ternary nanocomposites as probes. Graphene oxide was first modified with hemin and then with gold nanoflowers through an in situ growth method, which has significantly boosted the catalytic activity of this graphene oxide-based peroxidase mimetics. The biocatalytical activity of this ECL immunosensor was thoroughly investigated to achieve selective recognition of the analyte molecules (PSA) by specific binding between antigens and antibodies. The limit of detection was calculated to be 0.32 pg mL-1 with a signal-to-noise ratio of 3. A broad linear range from 7.5 × 10-4 to 2.5 ng mL-1 was obtained. Such step-by-step assembled biosensor showed controlled nanostructure and exhibited promising application in analysis of human serum samples with a recovery range of 90.6-111.8% and a RSD range of 3.9-5.5%.

In Situ Single-Molecule Imaging of MicroRNAs in Switchable Migrating Cells under Biomimetic Confinement.

Spatial imaging of RNAs in single cells is extremely charming for deciphering of regulatory mechanisms in multiple migration modes during tumor metastasis. Herein, enzyme-free-mediated cascade amplified nanoprobes were designed for in situ single-molecule imaging of dual-microRNAs (miRNAs) in switchable migrating cells. Differential expression and localization of dual-miRNAs were clearly exhibited in multiple cell lines attributed to enhanced sensitivity via the cascade signal amplification strategy. Significantly, in situ three-dimensional (3D) imaging of dual-miRNAs in transition of cell migration phenotypes was successfully reconstructed in both non-confined and confined microenvironments in vitro, of which differential spatial distribution was observed in a single cell. This is very promising for exploring key roles of spatial RNA distribution in migrating cells at the single-molecule level, which will advance revealing the molecular mechanism and physical principle in 3D cell migration in vivo.

Self-assembled plasmonic nanoarrays for enhanced bacterial identification and discrimination.

The rapid and accurate bacterial testing is a critical step for the management of infectious diseases, but challenges remain largely due to a lack of advanced sensing tools. Here we report the development of highly plasmon-active, biofunctional nanoparticle arrays for simultaneous capture, identification, and differentiation of bacteria by surface-enhanced Raman scattering (SERS). The nanoarrays were facilely prepared through an electrostatic mechanism-controlled self-assembly of metallic nanoparticles at liquid-liquid interfaces, and exhibited high SERS sensitivity beyond femtomole, good reproducibility (relative standard deviation of 2.7%) and stability. Modification of the nanoarrays with concanavalin A allowed to effective capture of both Gram-positive and Gram-negative bacteria (bacterial-capture efficiency maintained beyond 50%) at bacterial concentrations ranging from 50 to 2000 CFU mL-1, as determined by the plate-counting method. Moreover, single-cell Raman fingerprinting and discrimination of eight different bacteria species with high signal-to-noise ratio, excellent spectral reproducibility, and a total assay time of 1.5 h was achieved under fairly mild conditions (24 µW, acquisition time: 1 s). Collectively, we believe that our biofunctionalized, SERS-based self-assembled nanoarrays have great potential to help in rapid and label-free bacterial diagnosis and phenotyping study.

Proximity binding induced nucleic acid cascade amplification strategy for ultrasensitive homogeneous detection of PSA.

In this work, based on the powerful cycle amplification cascades of proximity hybridization-induced hybridization chain reaction and catalyzed hairpin assembly, we engineered a nonenzymatic and ultrasensitive method which combined the Mg2+-DNAzyme recycling signal amplification for the analysis of the human prostate specific antigen. Herein, we adopted PSA-conjugates as triggers in the self-assembly process of two hairpin DNAs (H1, H2) into the products of the CHA which could activate the HCR to induce repeated hybridization. And both ends of each adjacent sequence of the HCR products could form a unit of Mg2+-DNAzyme which in presence of cofactor Mg2+ could recognize and cyclically cleave the hairpin probes in the solution and thus generate observably enhanced fluorescent signal. Benefit from the nucleic acid circuit amplification strategy, PSA of concentration low to 0.73 pg mL-1 was detected in this system. This homogeneous sensing method in solution avoid the use of the sophisticated equipment and complex operation, as well as addition of artificial enzyme, thus greatly reducing the constraints and complexity of experimental conditions. Moreover, considering most protein biomarkers in serum don't have their corresponding aptamers, this sensing method provide a general sensing approach for homogeneous sensitive detection of these important protein biomarkers which transfer rough antigen-antibody interactivity to smart signal amplification sensing strategies, thus exhibiting a remarkable prospect in clinical application.

Single Biomolecule Imaging by Electrochemiluminescence.

Herein, a single biomolecule is imaged by electrochemiluminescence (ECL) using Ru(bpy)32+-doped silica/Au nanoparticles (RuDSNs/AuNPs) as the ECL nanoemitters. The ECL emission is confined to the local surface of RuDSNs leading to a significant enhancement in the intensity. To prove the concept, a single protein molecule at the electrode is initially visualized using the as-prepared RuDSN/AuNPs nanoemitters. Furthermore, the nanoemitter-labeled antibody is linked at the cellular membrane to image a single membrane protein at one cell, without the interference of current and optical background. The success in single-biomolecule ECL imaging solves the long-lasting task in the ultrasensitive ECL analysis, which should be able to provide more elegant information about the protein in cellular biology.

Highly efficient sub-nanometer RuxCuyP2 nanoclusters designed for hydrogen evolution under alkaline media.

Design of highly active and stable non-precious electrocatalysts towards hydrogen evolution reaction (HER) is a hot research topic in low cost, clean and sustainable hydrogen energy field, yet remaining the important challenge caused by the sluggish reaction kinetics for water-alkali electrolyzers. Herein, a robust electrocatalyst is proposed by designing a novel sub-nanometer of copper and ruthenium bimetallic phosphide nanoclusters (RuxCuyP2) supported on a graphited carbon nanofibers (CNF). Uniform RuxCuyP2 (~1.90 nm) on the surface of CNF are obtained by introducing the dispersed Ru, thereby improving the intrinsic activity for HER. On optimizing the Ru ratio, the (x = y = 1) RuCuP2/CNF catalyst exhibits an excellent HER electroactivity with an overpotential of 10 mV in 1.0 M NaOH electrolyte to produce 10 mA cm-2 current density, which is lower than commercial 20% Pt/C in alkaline solution. Moreover, the kinetic study demonstrated that electrochemical activation energies for HER of RuCuP2/CNF is 20.7 kJ mol-1 highest among different ratio bimetallic phosphide. This simple, cost-effective, and environmentally friendly methodology can pave the way for exploitation of bimetallic phosphide nanoclusters for catalyst design.

Dual-Mode SERS and Electrochemical Detection of miRNA Based on Popcorn-like Gold Nanofilms and Toehold-Mediated Strand Displacement Amplification Reaction.

MicroRNA (miRNA) has emerged as one of the ideal target biomarker analytes for cancer detection because its abnormal expression is closely related to the occurrence of many cancers. In this work, we combined three-dimensional (3D) popcorn-like gold nanofilms as novel surface-enhanced Raman scattering (SERS)-electrochemistry active substrates with toehold-mediated strand displacement reactions (TSDRs) to construct a DNA molecular machine for SERS-electrochemistry dual-mode detection of miRNA. 3D popcorn-like spatial structures generated more active "hot spots" and thus enhanced the sensitivity of SERS and electrochemical signals. Besides, the TSDRs showed high sequence-dependence and high specificity. The addition of target miRNA will trigger the molecular machine to perform two TSDRs in the presence of signal DNA strands modified by R6G (R6G-DNA), thus achieving an enzyme-free amplification detection of miRNA with a low limit of detection of 0.12 fM (for the SERS method) and 2.2 fM (for the electrochemical method). This biosensor can also serve as a universally amplified and sensitive detection platform for monitoring different biomarkers, such as cancer-related DNA, messenger RNA, or miRNA molecules, with high selectivity by changing the corresponding probe sequence.

Transpeptidation-mediated single-particle imaging assay for sensitive and specific detection of sortase with dark-field optical microscopy.

Transpeptidation of surface proteins catalyzed by the transpeptidase sortase plays a critical role in the infection process of Gram-positive pathogen. Monitoring sortase activity and screening its inhibitors are of great significance to fundamental understanding of the infection mechanism and pharmaceutical development. Herein, we developed a digital single-particle imaging method to quantify sortase A (SrtA) activity based on transpeptidation-mediated assembly and enumeration of gold nanoparticles (GNPs). The assay utilizes two peptide stands, in which one has the SrtA recognition sequence LPXTG motif while the other carries an oligoglycine nucleophile at the one end and a biotin group at the other. The presence of SrtA enables the ligation of two peptides and allows for the immobilization of streptavidin-functionalized GNPs. Thus, SrtA activity can be quantified by imaging and enumeration of the surface-assembled GNPs at the single-particle level via dark-field microscopy. The single-particle method was highly sensitive to SrtA activity with a low detection limit of 7.9 pM and a wide linear dynamic range from 0.05 to 50 nM. Besides detection of SrtA in complex biological samples such as Gram-positive pathogen lysates, the proposed method was also successfully applied to estimate the half-maximal inhibitory concentration (IC50) values of SrtA inhibitors (curcumin, berberine hydrochloride and quercetin). The present method, combining single-GNP counting and dark-field imaging, provides a facile and novel analytical tool for SrtA activity and its inhibitor screening.

An electrochemiluminescence sensor for 17ß-estradiol detection based on resonance energy transfer in α-FeOOH@CdS/Ag NCs.

An electrochemiluminescence (ECL) resonance energy transfer system is constructed for 17ß-estradiol (E2) detection using α-FeOOH@CdS nanospheres as the ECL-active substrates and Ag NCs as an efficient quencher. CdS QDs loaded onto three-dimensional (3D) urchin-like α-FeOOH nanospheres (α-FeOOH@CdS nanospheres) exhibited excellent ECL responses, which is attributed to dual-amplification of α-FeOOH frameworks. The 3D hierarchical structure of the α-FeOOH nanospheres provided abundant sites for loading ECL-active species, thus significantly improving the ECL performance of substrates; While Fe3+ presented on surface of α-FeOOH nanospheres could be reduced to Fe2+ in negative potentials, after which might activate persulfate in a Fenton-like process, resulting in more sulfate free radicals for more effective ECL responses via electron transfer reactions. Additionally, Ag nanoclusters (Ag NCs) stabilized by single stranded oligonucleotide were introduced as quenching probes for CdS QDs owing to the well-matched donor-acceptor spectrum for efficient energy transfer, which makes them appropriate for detection of E2. The proposed strategy displayed a desirable dynamic range from 0.01 to 10 pg mL-1 with a limit of detection of 0.003 pg mL-1. The proposed strategy based on the ECL-RET strategy offered an ideal way for E2 detection, and also revealed an alternative platform for detection of other small molecules.

Artificial Aquaporin That Restores Wound Healing of Impaired Cells.

Artificial aquaporins are synthetic molecules that mimic the structure and function of natural aquaporins (AQPs) in cell membranes. The development of artificial aquaporins would provide an alternative strategy for treatment of AQP-related diseases. In this report, an artificial aquaporin has been constructed from an amino-terminated tubular molecule, which operates in a unimolecular mechanism. The artificial channel can work in cell membranes with high water permeability and selectivity rivaling those of AQPs. Importantly, the channel can restore wound healing of the cells that contain function-lost AQPs.

Construction of Dual-Color Probes with Target-Triggered Signal Amplification for In Situ Single-Molecule Imaging of MicroRNA.

The in vitro detection of low abundance biomolecules via nonenzymatic signal amplification is an attractive strategy. However, it remains a challenge to monitor targets of interest in situ in living cells by low-background interference and visualized enzyme-free signal amplification strategies. Taking advantage of the single-molecule imaging and dynamic DNA nanotechnologies, we have achieved the target-triggered self-assembly of nanostructure-based dual-color fluorescent probes (NDFPs) by an enzyme-free toehold-mediated strand displacement cascade. NDFPs will facilitate the simple and visualized monitoring of microRNA (miRNA) at the femtomolar level. The recycled miRNA can be considered as the catalyst for the assembly of multiple H1/H2 duplexes. This generated the fluorescence signal of the enhanced target expression, indicating both in vitro and in vivo signal-amplified imaging. Moreover, the NDFPs improved the measurement accuracy by dual-color colocalization imaging to greatly avoid false-positive signals and enabled the successful in situ imaging of miRNA in living cells in real time. This work provides a strategy to visually monitor and study the integration of signal amplification detection and single-molecule imaging. NDFPs may be an important step toward the enzyme-free amplified monitoring and imaging of various biomolecules in living cells at the single-molecule level.