Application Of Small-molecule Fluorescent Probes In Super-Resolution Imaging

The cell membrane,the outermost layer of a cell,separates the cell from the environment.Many physiological activities of cells are controlled by the spatial organization of membrane molecules,such as the membrane carbohydrates and proteins Abnormal behavior of these molecules can lead to many diseases.Studying their distribution,size,and composition is critical for understanding how these molecules function in the biological and pathological processes.Direct observation at the molecular level is one of the best ways to know their distribution on cell membranes.As the size of these molecules is expected to lie below the diffraction limit,conventional fluorescence microscopy is clearly not appropriate.Recently,the super-resolution imaging technologies have broken the optical diffraction limit,and made tremendous contributions in the investigations of cell membranes,biological structures and molecular interactions.One of the most widely used techniques is direct stochastic optical reconstruction microscopy(dSTORM)because of its outstanding spatial resolution and convenient operation.Generally,one super-resolution image is reconstructed by obtaining enough localizations within a certain period of time.So the quality of the super-resolution image is related to the quality of obtained localizations,such as localization density and accuracy,which are directly affected by the nature of labeling probes.Therefore,to obtain a more precise super-resolution image,an accurate and complete labeling method is required.The ideal labeling probe should be small-sized,monovalent,high specificity and less interference with the target.Traditional labeling molecules with relatively large size and multivalent states,such as antibodies and lectins,have potential steric hindrance and cross-linking when recognizing target molecules.Fortunately,the emergence of some small-molecule probes,which are smaller and more flexible,has provided us an unprecedented choice.Therefore,we used dSTORM imaging combined with small-molecule fluorescent probes to study the distribution pattern of several important membrane molecules.The main results obtained are as follows:1.Carbohydrates are one of the most important components on the cell membrane.They participate in various physiological activities,and their aberrant expression is a consequence of pathological changes.In previous studies,carbohydrate analysis basically relied on lectins.However,discrimination between lectins still exists due to their multivalent character.Herein,an aptamer recognition method with high precise localization was developed for imaging membrane-bound N-acetylgalactosamine(GalNAc).By using dSTORM,we compared this aptamer recognition method with the lectin recognition method for observing the detailed structure of GalNAc at the nanometer scale.The results indicated that GalNAc formed irregular clusters on the cell membrane with a resolution of 23 ± 7 nm by aptamer recognition.Additionally,when treated with N-acetylgalactosidase,the aptamer-recognized GalNAc showed a more significant decrease in cluster size and localization density,thus verifying better specificity of aptamers than lectins.Collectively,our study suggested that aptamers could act as perfect substitutes for lectins in carbohydrate labeling,which would be of great potential value in the field of super-resolution fluorescence imaging.2.Globo H,as one of the most crucial cancer-associated carbohydrates,is exclusively overexpressed in a variety of cancers.However,the accurate localization and detailed morphology of globo H at the molecular level remain unclear.Here,we applied dSTORM imaging and relied on fluorophore-conjugated aptamers to solve the problem.The results showed that globo H organized as clusters on cell membranes with irregular shapes and different sizes.Significantly,globo H was found to have a higher expression level and larger clusters on various cancer cells than on non-cancer cells,which hinted that its specific distribution could be utilized for cancer diagnosis.Moreover,dual-color dSTORM imaging revealed the colocalization of globo H and other cancer-associated carbohydrates,and the clustering of globo H could be disrupted by the treatment of corresponding glycosidases,which indicated that these carbohydrates might intertwine in spatial organization and function cooperatively in cancers.Our work clarified the clustered distribution of globo H at the nanometer scale and revealed the potential interactions between cancer-associated carbohydrates,which paves the way for further understanding the relationship between the spatial structures and functions of carbohydrates in cancers.3.Epithelial cell adhesion molecule(EpCAM)is an important type I transmembrane protein that is overexpressed on the surfaces of most cancer cells and involved in various biological processes such as cell adhesion and cell signaling.Although it plays crucial roles in cell functions and tumorigenesis,questions concerning the detailed morphology,molecular stoichiometry,and the assembly mechanisms of EpCAM on cell membranes have not been fully elucidated.Here,we used fluorophore-conjugated peptides to recognize the EpCAM and quantitatively analyze the assembly pattern by dSTORM imaging.EpCAM was found to organize heterogeneous clusters that had different sizes and different numbers of EpCAM molecules on MCF-7 cell membranes.Moreover,dual-color dSTORM imaging revealed a significant correlation between EpCAM and tetraspanin CD9,and part of the EpCAM clusters could be disrupted by knockdown of CD9,which indicated that EpCAM might localize in tetraspaninenriched microdomains(TEMs)and function cooperatively with CD9 on cell membranes.In addition,the assembly of the membrane EpCAM was found to be limited by both cytoskeleton and glycosylation.Overall,our work clarified the clustered distribution of EpCAM and revealed the potential mechanisms of its clustering at the molecular level,promoting a deeper understanding of the nano-organization of membrane proteins.4.Nucleolin(NCL)is a multifunctional protein that mainly localizes in the nucleolus and also distributes in the nucleoplasm,cytoplasm and cell membrane.Most studies focus on its biofunctions in cell activities and diseases,however,its detailed distribution and organization pattern in situ remains obscure.Moreover,antibodies were commonly used to investigate NCL in cells.It is worth noting that antibody labeling of intracellular proteins needs detergents to permeabilize the membrane,which could disrupt the membrane structure and proteins.The emergence of aptamer AS1411 has provided us a good choice to recognize the NCL without permeabilization owing to its superior cellular uptake and enhanced stability.Therefore,we applied aptamer AS 1411 to super-resolution imaging to investigate the distribution of NCL at a nanometer level.Aptamer achieved a better recognition of intracellular NCL and displayed the detailed structure of NCL in different parts of cells.Significantly,cytoplasmic and membrane NCL had higher expression and larger clusters in cancer cells than that in normal cells.Our work presented a detailed organization of NCL in cells and revealed the distribution differences between cancer cells and normal cells,which promotes the understanding of its functions in physiology and pathology.5.The prostate-specific membrane antigen(PSMA)is an important tumor antigen that is highly expressed in prostate cancer cells.It not only participates in diverse cell activities but also functions like enzymes.Insights into the distribution of PSMA would promote the understanding of its roles in biological process.Based on its enzymatic activity,we constituted an inhibitor-based probe for dSTORM imaging of PSMA with high resolution.The small-sized inhibitor probe had recognized more PSMA and showed a weak linkage error of membrane proteins than antibody on cell membranes.Relying on this probe,we found that PSMA organized into clusters with different sizes.Remarkably,dual-color dSTORM highlighted the significant colocalization of PSMA and folate receptor,which hinted that these two proteins might function cooperatively in folate transport.Our work verified that the clustered distribution of PSMA at the nanometer scale and revealed the potential interactions between PSMA and folate receptor,which is helpful for understanding the biological function of PSMA.Notably,this inhibitor-based probe renders it a versatile tool of choice for many more targets amenable to dSTORM imaging.