Comprehensive integration of single-cell transcriptomic data illuminates the regulatory network architecture of plant cell fate specification

Plant morphogenesis relies on precise gene expression programs at the proper time and position which is orchestrated by transcription factors(TFs)in intricate regulatory networks in a cell-type specific manner.Here we introduced a comprehensive single-cell transcriptomic atlas of Arabidopsis seedlings.This atlas is the result of meticulous integration of 63 previously published scRNA-seq datasets,addressing batch effects and conserving biological variance.This integration spans a broad spectrum of tissues,including both below-and above-ground parts.Utilizing a rigorous approach for cell type annotation,we identified 47 distinct cell types or states,largely expanding our current view of plant cell compositions.We systematically constructed cell-type specific gene regulatory networks and uncovered key regulators that act in a coordinated manner to control cell-type specific gene expression.Taken together,our study not only offers extensive plant cell atlas exploration that serves as a valuable resource,but also provides molecular insights into gene-regulatory programs that varies from different cell types.

Single-cell RNA sequencing in cornea research:Insights into limbal stem cells and their niche regulation

The corneal epithelium is composed of stratified squamous epithelial cells on the outer surface of the eye,which acts as a protective barrier and is critical for clear and stable vision.Its continuous renewal or wound healing depends on the proliferation and differentiation of limbal stem cells(LSCs),a cell population that resides at the limbus in a highly regulated niche.Dysfunction of LSCs or their niche can cause limbal stem cell deficiency,a disease that is manifested by failed epithelial wound healing or even blindness.Nevertheless,compared to stem cells in other tissues,little is known about the LSCs and their niche.With the advent of single-cell RNA sequencing,our understanding of LSC characteristics and their microenvironment has grown considerably.In this review,we summarized the current findings from single-cell studies in the field of cornea research and focused on important advancements driven by this technology,including the heterogeneity of the LSC population,novel LSC markers and regulation of the LSC niche,which will provide a reference for clinical issues such as corneal epithelial wound healing,ocular surface reconstruction and interventions for related diseases.

Observing single cells in whole organs with optical imaging

Cells are the basic unit of human organs that are not fully understood.The revolutionary advancements of optical imaging alowed us to observe single cells in whole organs,revealing the complicated composition of cells with spatial information.Therefore,in this review,we revisit the principles of optical contrast related to those biomolecules and the optical techniques that transform optical contrast into detectable optical signals.Then,we describe optical imaging to achieve threedimensional spatial discrimination for biological tisutes.Due to the milky appearance of tissues,the spatial information burred deep in the whole organ.Fortunately,strategies developed in the last decade could circumvent this issue and lead us into a new era of investigation of the cells with their original spatial information.

Single-cell transcriptomic dissection of the cellular and molecular events underlying the triclosan-induced liver fibrosis in mice

Background: Triclosan [5-chloro-2-(2,4-dichlorophenoxy) phenol, TCS], a common antimicrobial additive in many personal care and health care products, is frequently detected in human blood and urine. Therefore, it has been considered an emerging and potentially toxic pollutant in recent years. Long-term exposure to TCS has been suggested to exert endocrine disruption effects, and promote liver fibrogenesis and tumorigenesis. This study was aimed at clarifying the underlying cellular and molecular mechanisms of hepatotoxicity effect of TCS at the initiation stage.Methods: C57BL/6 mice were exposed to different dosages of TCS for 2 weeks and the organ toxicity was evaluated by various measurements including complete blood count, histological analysis and TCS quantification. Single cell RNA sequencing(scRNA-seq) was then carried out on TCS-or mock-treated mice livers to delineate the TCS-induced hepatotoxicity. The acquired single-cell transcriptomic data were analyzed from different aspects including differential gene expression, transcription factor(TF) regulatory network, pseudotime trajectory, and cellular communication, to systematically dissect the cellular and molecular events after TCS exposure. To verify the TCS-induced liver fibrosis,the expression levels of key fibrogenic proteins were examined by Western blotting, immunofluorescence, Masson’s trichrome and Sirius red stainings. In addition, normal hepatocyte cell MIHA and hepatic stellate cell LX-2 were used as in vitro cell models to experimentally validate the effects of TCS by immunological, proteomic and metabolomic technologies.Results: We established a relatively short term TCS exposure murine model and found the TCS mainly accumulated in the liver. The scRNA-seq performed on the livers of the TCS-treated and control groups profiled the gene expressions of > 76,000 cells belonging to 13 major cell types. Among these types, hepatocytes and hepatic stellate cells(HSCs)were significantly increased in TCS-treated group. We found that TCS promoted fibrosis-associated proliferation of hepatocytes, in which Gata2 and Mef2c are the key driving TFs. Our data also suggested that TCS induced the proliferation and activation of HSCs, which was experimentally verified in both liver tissue and cell model. In addition,other changes including the dysfunction and capillarization of endothelial cells, an increase of fibrotic characteristics in B plasma cells, and M2 phenotype-skewing of macrophage cells, were also deduced from the scRNA-seq analysis, and these changes are likely to contribute to the progression of liver fibrosis. Lastly, the key differential ligand-receptor pairs involved in cellular communications were identified and we confirmed the role of GAS6_AXL interactionmediated cellular communication in promoting liver fibrosis.Conclusions: TCS modulates the cellular activities and fates of several specific cell types(including hepatocytes, HSCs,endothelial cells, B cells, Kupffer cells and liver capsular macrophages) in the liver, and regulates the ligand-receptor interactions between these cells, thereby promoting the proliferation and activation of HSCs, leading to liver fibrosis.Overall, we provide the first comprehensive single-cell atlas of mice livers in response to TCS and delineate the key cellular and molecular processes involved in TCS-induced hepatotoxicity and fibrosis.

Tunable microfluidic chip for single-cell deformation study

Microfluidic phenotyping methods have been of vital importance for cellular characterization,especially for evaluating single cells.In order to study the deformability of a single cell,we devised and tested a tunable microfluidic chip-based method.A pneumatic polymer polydimethylsiloxane(PDMS)membrane was designed and fabricated abutting a single-cell trapping structure,so the cell could be squeezed controllably in a lateral direction.Cell contour changes under increasing pressure were recorded,enabling the deformation degree of different types of single cell to be analyzed and compared using computer vision.This provides a new perspective for studying mechanical properties of cells at the single cell level.

Single cell RNA sequencing reveals mesenchymal heterogeneity and critical functions of Cd271 in tooth development

BACKGROUND Accumulating evidence suggests that the maxillary process,to which cranial crest cells migrate,is essential to tooth development.Emerging studies indicate that Cd271 plays an essential role in odontogenesis.However,the underlying mechanisms have yet to be elucidated.AIM To establish the functionally heterogeneous population in the maxillary process,elucidate the effects of Cd271 deficiency on gene expression differences.METHODS p75NTR knockout(Cd271-/-)mice(from American Jackson laboratory)were used to collect the maxillofacial process tissue of p75NTR knockout mice,and the wildtype maxillofacial process of the same pregnant mouse wild was used as control.After single cell suspension,the cDNA was prepared by loading the single cell suspension into the 10x Genomics Chromium system to be sequenced by NovaSeq6000 sequencing system.Finally,the sequencing data in Fastq format were obtained.The FastQC software is used to evaluate the quality of data and CellRanger analyzed the data.The gene expression matrix is read by R software,and Seurat is used to control and standardize the data,reduce the dimension and cluster.We search for marker genes for subgroup annotation by consulting literature and database;explore the effect of p75NTR knockout on mesenchymal stem cells(MSCs)gene expression and cell proportion by cell subgrouping,differential gene analysis,enrichment analysis and protein-protein interaction network analysis;understand the interaction between MSCs cells and the differentiation trajectory and gene change characteristics of p75NTR knockout MSCs by cell communication analysis and pseudo-time analysis.Last we verified the findings single cell sequencing in vitro.RESULTS We identified 21 cell clusters,and we re-clustered these into three subclusters.Importantly,we revealed the cell–cell communication networks between clusters.We clarified that Cd271 was significantly associated with the regulation of mineralization.CONCLUSION This study provides comprehensive mechanistic insights into the maxillary-process-derived MSCs and demonstrates that Cd271 is significantly associated with the odontogenesis in mesenchymal populations.

A SARS-CoV-2 neutralizing antibody discovery by single cell sequencing and molecular modeling

Although vaccines have been developed,mutations of SARS-CoV-2,especially the dominant B.1.617.2(delta)and B.1.529(omicron)strains with more than 30 mutations on their spike protein,have caused a significant decline in prophylaxis,calling for the need for drug improvement.Antibodies are drugs preferentially used in infectious diseases and are easy to get from immunized organisms.The current study combined molecular modeling and single memory B cell sequencing to assess candidate sequences before experiments,providing a strategy for the fabrication of SARS-CoV-2 neutralizing antibodies.A total of 128 sequences were obtained after sequencing 196 memory B cells,and 42 sequences were left after merging extremely similar ones and discarding incomplete ones,followed by homology modeling of the antibody variable region.Thirteen candidate sequences were expressed,of which three were tested positive for receptor binding domain recognition but only one was confirmed as having broad neutralization against several SARS-CoV-2 variants.The current study successfully obtained a SARS-CoV-2 antibody with broad neutralizing abilities and provided a strategy for antibody development in emerging infectious diseases using single memory B cell BCR sequencing and computer assistance in antibody fabrication.

Application of single-cell omics in inflammatory bowel disease

Over the past decade,the advent of single cell RNA-sequencing has revolutionized the approach in cellular transcriptomics research.The current technology offers an unbiased platform to understand how genotype correlates to phenotype.Single-cell omics applications in gastrointestinal(GI)research namely inflammatory bowel disease(IBD)has become popular in the last few years with multiple publications as single-cell omics techniques can be applied directly to the target organ,the GI tract at the tissue level.Through examination of mucosal tissue and peripheral blood in IBD,the recent boom in single cell research has identified a myriad of key immune players from enterocytes to tissue resident memory T cells,and explored functional heterogeneity within cellular subsets previously unreported.As we begin to unravel the complex mucosal immune system in states of health and disease like IBD,the power of exploration through single-cell omics can change our approach to translational research.As novel techniques evolve through multiplexing single-cell omics and spatial transcriptomics come to the forefront,we can begin to fully comprehend the disease IBD and better design targets of treatment.In addition,hopefully these techniques can ultimately begin to identify biomarkers of therapeutic response and answer clinically relevant questions in how to tailor individual therapy to patients through personalized medicine.

Alzheimer’s disease, neural stem cells and neurogenesis: cellular phase at single-cell level

Alzheimer’s disease cannot be cured as of yet.Our current understanding on the causes of Alzheimer’s disease is limited.To develop treatments,experimental models that represent a particular cellular phase of the disease and more rigorous scrutiny of the cellular pathological mechanisms are crucial.In recent years,Alzheimer’s disease research underwent a paradigm shift.According to this tendency,Alzheimer’s disease is increasingly being conceived of a disease where not only neurons but also multiple cell types synchronously partake to manifest the pathology.Knowledge on every cell type adds an alternative approach and hope for the efforts towards the treatment.Neural stem cells and their neurogenic ability are making an appearance as a new aspect of the disease manifestation based on the recent findings that neurogenesis reduces dramatically in Alzheimer’s disease patients compared to healthy individuals.Therefore,understanding how neural stem cells can form new neurons in Alzheimer’s disease brains holds an immense potential for clinics.However,this provocative idea requires further evidence and tools for investigation.Recently,single cell sequencing appeared as a revolutionary tool to understand cellular programs in unprecedented resolution and it will undoubtedly facilitate comprehensive investigation of different cell types in Alzheimer’s disease.In this mini-review,we will touch upon recent studies that use single cell sequencing for investigating cellular response in Alzheimer’s disease and some consideration pertaining to the utilization of neural regeneration for Alzheimer’s disease research.

Applications of single cell RNA sequencing to research of stem cells

Stem cells(SCs)with their self-renewal and pluripotent differentiation potential,show great promise for therapeutic applications to some refractory diseases such as stroke,Parkinsonism,myocardial infarction,and diabetes.Furthermore,as seed cells in tissue engineering,SCs have been applied widely to tissue and organ regeneration.However,previous studies have shown that SCs are heterogeneous and consist of many cell subpopulations.Owing to this heterogeneity of cell states,gene expression is highly diverse between cells even within a single tissue,making precise identification and analysis of biological properties difficult,which hinders their further research and applications.Therefore,a defined understanding of the heterogeneity is a key to research of SCs.Traditional ensemble-based sequencing approaches,such as microarrays,reflect an average of expression levels across a large population,which overlook unique biological behaviors of individual cells,conceal cell-to-cell variations,and cannot understand the heterogeneity of SCs radically.The development of high throughput single cell RNA sequencing(scRNA-seq)has provided a new research tool in biology,ranging from identification of novel cell types and exploration of cell markers to the analysis of gene expression and predicating developmental trajectories.scRNA-seq has profoundly changed our understanding of a series of biological phenomena.Currently,it has been used in research of SCs in many fields,particularly for the research of heterogeneity and cell subpopulations in early embryonic development.In this review,we focus on the scRNA-seq technique and its applications to research of SCs.

Hierarchically mesoporous carbon spheres coated with a single atomic Fe-N-C layer for balancing activity and mass transfer in fuel cells

Novel cost-effective fuel cells have become more attractive due to the demands for rare and expensive platinum-group metal(PGM)catalysts for mitigating the sluggish kinetics of the oxygen reduction reaction(ORR).The high-cost PGM catalyst in fuel cells can be replaced by earth-abundant transition-metalbased catalysts,that is,an Fe-N-C catalyst,which is considered one of the most promising alternatives.However,the performance of the Fe-N-C catalyst is hindered by the low catalytic activity and poor stability,which is caused by insufficient active sites and the lack of optimization of the triple-phase interface for mass transportation.Herein,a novel Fe–N–C catalyst consisting of mono-dispersed hierarchically mesoporous carbon sphere cores and single Fe atom-dispersed functional shells are presented.The synergistic effect between highly dispersed Fe-active sites and well-organized porous structures yields the combination of high ORR activity and high mass transfer performance.The half-wave potential of the catalyst in 0.1M H_(2)SO_(4) is 0.82 V versus reversible hydrogen electrode,and the peak power density is 812 mW·cm^(−2) in H_(2)–O_(2) fuel cells.Furthermore,it shows superior methanol tolerance,which is almost immune to methanol poisoning and generates up to 162 mW·cm^(−2) power density in direct methanol fuel cells.

Migratory properties of cultured olfactory ensheathing cells by single-cell migration assay

嗅觉的 ensheathing 房间(OEC ) 是有 axonal 的 glial 房间的一种唯一的类型支持生长的性质。OEC 移植作为 axonal 损害和 demyelinating 疾病的有希望的试验性的治疗出现了。然而， OEC 的一些基本细胞的性质仍然保持不清楚。在这研究，我们发现不同 OEC 候补选手人口基于单个孤立的房间的微速摄影的成像展出了不同迁移的性质，可能由于他们的不同细胞骨架组织。而且， OEC 潜水艇人口在单个房间的迁居试金显示了不同吸引人的迁移的回答到 lysophosphatidic 酸(LPA ) 的一个坡度。最后，我们发现了人口自发地转变了成对方的那个 OEC 代用品。一起，这些结果示威，第一次到我们的知识，不同 OEC 潜水艇人口显示不同迁移的性质试管内并且提供新证据当一个单个房间与韧性的功能的显型打字，支持 OEC 的观点。

Single-cell analyses of circulating tumor cells

Circulating tumor cells(CTCs) are a population of tumor cells mediating metastasis, which results in most of the cancer related deaths. The number of CTCs in the peripheral blood of patients is rare, and many platforms have been launched for detection and enrichment of CTCs. Enumeration of CTCs has already been used as a prognosis marker predicting the survival rate of cancer patients. Yet CTCs should be more potential. Studies on CTCs at single cell level may help revealing the underlying mechanism of tumorigenesis and metastasis. Though far from developed, this area of study holds much promise in providing new clinical application and deep understanding towards metastasis and cancer development.

Single-cell analysis of tumors:Creating new value for molecular biomarker discovery of cancer stem cells and tumor-infiltrating immune cells

Biomarker-driven individualized treatment in oncology has made tremendous progress through technological developments, new therapeutic modalities and a deeper understanding of the molecular biology for tumors, cancer stem cells and tumor-infiltrating immune cells. Recent technical developments have led to the establishment of a variety of cancer-related diagnostic, prognostic and predictive biomarkers. In this regard, different modern OMICs approaches were assessed in order to categorize and classify prognostically different forms of neoplasia. Despite those technical advancements, the extent of molecular heterogeneity at the individual cell level in human tumors remains largely uncharacterized. Each tumor consists of a mixture of heterogeneous cell types. Therefore, it is important to quantify the dynamic cellular variations in order to predict clinical parameters, such as a response to treatment and or potential for disease recurrence. Recently, single-cell based methods have been developed to characterize the heterogeneity in seemingly homogenous cancer cell populations prior to and during treatment. In this review, we highlight the recent advances for single-cell analysis and discuss the challenges and prospects for molecular characterization of cancer cells, cancer stem cells and tumor-infiltrating immune cells.

Research on the boron contamination at the p/i interface of microcrystalline silicon solar cells deposited in a single PECVD chamber

This paper studies boron contamination at the interface between the p and i layers of μc-Si：H solar cells deposited in a single-chamber PECVD system. The boron depth profile in the i layer was measured by Secondary Ion Mass Spectroscopy. It is found that the mixed-phase μc-Si：H materials with 40% crystalline volume fraction is easy to be affected by the residual boron in the reactor. The experimental results showed that a 500-nm thick μc-Si：H covering layer or a 30-seconds of hydrogen plasma treatment can effectively reduce the boron contamination at the p/i interface. However, from viewpoint of cost reduction, the hydrogen plasma treatment is desirable for solar cell manufacture because the substrate is not moved during the hydrogen plasma treatment.

Reduction of the phosphorus contamination for plasma deposition of p-i-n microcrystalline silicon solar cells in a single chamber

This paper investigates several pretreatment techniques used to reduce the phosphorus contamination between solar cells. They include hydrogen plasma pretreatment, deposition of a p-type doped layer, i-a-Si：H or μc-Si：H covering layer between solar cells. Their effectiveness for the pretreatment is evaluated by means of phosphorus concentration in films, the dark conductivity of p-layer properties and cell performance.

Micromorph tandem solar cells: optimization of the microcrystalline silicon bottom cell in a single chamber system

We report on the development of single chamber deposition of microcrystalline and micromorph tandem solar cells directly onto low-cost glass substrates. The cells have pin single-junction or pin/pin double-junction structures on glass substrates coated with a transparent conductive oxide layer such as SnO2 or ZnO. By controlling boron and phosphorus contaminations, a single-junction microcrystalline silicon cell with a conversion efficiency of 7.47% is achieved with an i-layer thickness of 1.2 μm. In tandem devices, by thickness optimization of the microcrystalline silicon bottom solar cell, we obtained an initial conversion efficiency of 9.91% with an aluminum （Al） back reflector without a dielectric layer. In order to enhance the performance of the tandem solar cells, an improved light trapping structure with a ZnO/Al back reflector is used. As a result, a tandem solar cell with 11.04% of initial conversion efficiency has been obtained.

Identifying EGFR-Expressed Cells and Detecting EGFR Multi-Mutations at Single-Cell Level by Microfluidic Chip

EGFR mutations companion diagnostics have been proved to be crucial for the efficacy of tyrosine kinase inhibitor targeted cancer therapies. To uncover multiple mutations occurred in minority of EGFR-mutated cells,which may be covered by the noises from majority of unmutated cells, is currently becoming an urgent clinical requirement. Here we present the validation of a microfluidic-chip-based method for detecting EGFR multimutations at single-cell level. By trapping and immunofluorescently imaging single cells in specifically designed silicon microwells, the EGFR-expressed cellswere easily identified. By in situ lysing single cells, the cell lysates of EGFR-expressed cells were retrieved without cross-contamination. Benefited from excluding the noise from cells without EGFR expression, the simple and cost-effective Sanger's sequencing, but not the expensive deep sequencing of the whole cell population, was used to discover multi-mutations. We verified the new method with precisely discovering three most important EGFR drugrelated mutations from a sample in which EGFR-mutated cells only account for a small percentage of whole cell population. The microfluidic chip is capable of discovering not only the existence of specific EGFR multi-mutations,but also other valuable single-cell-level information: on which specific cells the mutations occurred, or whether different mutations coexist on the same cells. This microfluidic chip constitutes a promising method to promote simple and cost-effective Sanger's sequencing to be a routine test before performing targeted cancer therapy.

Conformationally Dynamic Supramolecular Single Chain Nanogels for Mutilscale Biomechanical Regulation of Stem Cells

Objective The binding of cell adhesive peptides(such as RGD)to integrins initiates the recruitment of cytoplasmic adaptor proteins(e.g.,vinculin)and the formation of focal adhesion(FA)complexes required for cell adhesion.The ability to manipulate this ligand-mediated cell adhesion process is crucial for regulating cell migration,cell differentiation,injury healing,and immune response.Some recent studies reported the importance of the tether length/mobility of the cell adhesive ligands in regulating the traction force development of cells.In the native cellular microenvironment,such a dynamic change in the nanoscale tether length of bioactive ligands is often mediated by conformational changes of the structural proteins due to protein folding or degradation.However,no prior studies have demonstrated the modulation of the ligand tether mobility by controlling the intramolecular folding of polymeric linkers.Unfoldable synthetic macromolecules with easy synthetic routes and controllable structures,such as supramolecular host-guest single chain nanogels(SCNGs),are ideal candidates for mimicking the changes in the tether mobility of bioactive ligands via biorthogonal triggers.Methods S,S’-bis(a’a’-dimethyl-a’’-propargyl acetate)trithiocarbonate was first used to mediate the RAFT polymerization of N,N-dimethyl acrylamide,vinyl-adamantane and vinyl-β-cyclodextrin to yield the ADA@CD-SCNGs.The preparation of the unfoldable host-guest SCNGs was evidenced by the by gel permeation chromatography,proton nuclear magnetic resonance spectroscopy,atomic force microscopy and dynamic light scattering.Then the RGD peptide was conjugated to the alkynyl group on one end of the SCNGs before immobilizing the material on the substrate,which was confirmed by scanning electron microscopy(SEM).The regulation of cell behaviours by unfolding of the SCNG-RGD was confirmed by immunofluorescence staining of vinculin and Yes-associated protein(YAP).Results The preparation of ADA@CD-SCNGs was confirmed by GPC which showed a unimodal molecular weight distribution.DLS and AFM data also proved that the SCNGs had an average diameter of 12±3nm.SEM images showed that SCNGs were conjugated as a linker of RGD peptide to thiolated glass substrate at an average density of 162±11 particles/μm2.These particles disappeared after adding free competitive ADA guest molecules,indicating the triggered unfolding of the tether SCNGs.In addition,the unfolding of supramolecular ADA@CD-SCNGs was also evidenced by a decrease in the GPC elution time and a slight increase in the apparent molecular weight.These results show that the immobilized ADA@CD-SCNGs can be unfolded to tune the tether length and mobility of the conjugated RGD ligands.Then we investigated the regulation of the cell behaviors on the substrate by triggering the unfolding of SCNG linkers.A critical level of traction force is required to effectively initiate and maintain integrin-mediated formation of FA complexes and subsequent mechano-transduction signaling.An increased tether length in cell-adhesive ligands can lead to a diminished cell traction force as if cells are adhering to soft substrates.Here,the unfolding of the ADA@CD-SCNG-RGD triggered by the addition of free ADA led to disassembly of the mature focal adhesions in the cells as evidenced by the reduced vinculin and F-actin in staining.Subsequently,nuclear YAP also decreased significantly because of the impaired mechano-sensing and diminished cell cytoskeleton tension.In addition,the extensively spread cells gradually became round after the medium was supplemented with free competitive ADA to unfold the SCNG linker.These finding demonstrates that the substrates with the unfolded ADA@CD-SCNG-RGD only supported weak cell adhesions.In contrast,on the substrate conjugated with the nonunfoldable MBA-SCNG-RGD linker,the addition of free ADA resulted in no change in the spread cell morphology and protein expressions.These results indicate that the unfoldable host-guest ADA@CD-SCNG can be used to manipulate the nanoscale presentation of ligands to regulate cell behaviors.Conclusions We demonstrate the application of SCNGs as the supramolecular linker to tune the nanoscale ligand tether length.These findings demonstrate that the strategy of manipulating the tether mobility of bioactive ligands by using supramolecular SCNGs as linkers provides a highly tunable,biomimetic,and bio-orthogonal approach to study the dynamic events of cell adhesion.

Real-Time analysis of exosome secretion of single cells with single molecule imaging

The exosome-mediated response can promote or restrain the diseases by regulating the intracellular pathways,making the exosome become an effective marker for diagnosis and therapeutic control at the single-cell level.However,real-time analysis is hard to be achieved with traditional approaches because the exosomes usually need to be enriched by ultracentrifugation for a measurable signal-to-noise ratio.Recently developed label-free single-molecule imaging approaches may become an real-time quantitative tool for the analysis of single exosomes and related secretion behaviors of single living cells owing to their extreme sensitivity.