We present a powerful method for direct mRNA detection based on ligation-based recognition and in situ amplification, capable of single-cell imaging mRNA at single-nucleotide and single-molecule resolution. Attributed to the use of Splint R ligase that can ligate padlock probe with RNA as target template, this method can efficiently detect mRNA in the absence of reverse transcription. This method enables spatial localization and correlation analysis of gene expression in single cells, which helps us to elucidate gene function and regulatory mechanisms.

Electrochemiluminescence (ECL) is rapidly evolving from an analytical method into an optical microscopy. The orthogonality of the electrochemical trigger and the optical readout distinguishes it from classic microscopy and electrochemical techniques, owing to its near-zero background, remarkable sensitivity, and absence of photobleaching and phototoxicity. In this minireview, we summarize the recent advances in ECL imaging technology, emphasizing original configurations which enable the imaging of biological entities and the improvement of the analytical properties by increasing the complexity and multiplexing of bioassays. Additionally, mapping the (electro)chemical reactivity in space provides valuable information on nanomaterials and facilitates deciphering ECL mechanisms for improving their performances in diagnostics and (electro)catalysis. Finally, we highlight the recent achievements in imaging at the ultimate limits of single molecules, single photons or single chemical reactions, and the current challenges to translate the ECL imaging advances to other fields such as material science, catalysis and biology.

Plasma membrane (PM)-associated abscisic acid (ABA) signal transduction is an important component of ABA signaling. The C2-domain ABA-related (CAR) proteins have been reported to play a crucial role in recruiting ABA receptor PYR1/PYL/RCAR (PYLs) to the PM. However, the molecular details of the involvement of CAR proteins in membrane-delimited ABA signal transduction remain unclear. For instance, where this response process takes place and whether any additional members besides PYL are taking part in this signaling process. Here, the GUS-tagged materials for all Arabidopsis CAR members were used to comprehensively visualize the extensive expression patterns of the CAR family genes. Based on the representativeness of CAR1 in response to ABA, we determined to use it as a target to study the function of CAR proteins in PM-associated ABA signaling. Single-particle tracking showed that ABA affected the spatiotemporal dynamics of CAR1. The presence of ABA prolonged the dwell time of CAR1 on the membrane and showed faster lateral mobility. Surprisingly, we verified that CAR1 could directly recruit hypersensitive to ABA1 (HAB1) and SNF1-related protein kinase 2.2 (SnRK2.2) to the PM at both the bulk and single-molecule levels. Furthermore, PM localization of CAR1 was demonstrated to be related to membrane microdomains. Collectively, our study revealed that CARs recruited the three main components of ABA signaling to the PM to respond positively to ABA. This study deepens our understanding of ABA signal transduction.

Single-molecule fluorescence in situ hybridization (smFISH) represents a promising approach for the quantitative analysis of nucleic acid biomarkers in clinical tissue samples. However, low signal intensity and high background noise are complications that arise from diagnostic pathology when performed with smFISH-based RNA imaging in formalin-fixed paraffin-embedded (FFPE) tissue specimens. Moreover, the associated complex procedures can produce uncertain results and poor image quality. Herein, by combining the high specificity of split DNA probes with the high signal readout of ZnCdSe/ZnS quantum dot (QD) labeling, we introduce QD split-FISH, a high-brightness smFISH technology, to quantify the expression of mRNA in both cell lines and clinical FFPE tissue samples of breast cancer and lung squamous carcinoma. Owing to its high signal-to-noise ratio, QD split-FISH is a fast, inexpensive, and sensitive method for quantifying mRNA expression in FFPE tumor tissues, making it suitable for biomarker imaging and diagnostic pathology.

G-quadruplex (G4) is a four-stranded noncanonical DNA structure that has long been recognized as a potential hindrance to DNA replication. However, how replisomes effectively deal with G4s to avoid replication failure is still obscure. Here, using single-molecule and ensemble approaches, the consequence of the collision between bacteriophage T7 replisome and an intramolecular G4 located on either the leading or lagging strand is examined. It is found that the adjacent fork junctions induced by G4 formation incur the binding of T7 DNA polymerase (DNAP). In addition to G4, these inactive DNAPs present insuperable obstacles, impeding the progression of DNA synthesis. Nevertheless, T7 helicase can dismantle them and resolve lagging-strand G4s, paving the way for the advancement of the replication fork. Moreover, with the assistance of the single-stranded DNA binding protein (SSB) gp2.5, T7 helicase is also capable of maintaining a leading-strand G4 structure in an unfolded state, allowing for a fraction of T7 DNAPs to synthesize through without collapse. These findings broaden the functional repertoire of a replicative helicase and underscore the inherent G4 tolerance of a replisome.

α-synuclein (α-Syn) is a presynaptic protein that is involved in Parkinson\'s and other neurodegenerative diseases and binds to negatively charged phospholipids. Previously, we reported that α-Syn clusters synthetic proteoliposomes that mimic synaptic vesicles. This vesicle-clustering activity depends on a specific interaction of α-Syn with anionic phospholipids. Here, we report that α-Syn surprisingly also interacts with the neutral phospholipid lysophosphatidylcholine (lysoPC). Even in the absence of anionic lipids, lysoPC facilitates α-Syn-induced vesicle clustering but has no effect on Ca2+-triggered fusion in a single vesicle-vesicle fusion assay. The A30P mutant of α-Syn that causes familial Parkinson disease has a reduced affinity to lysoPC and does not induce vesicle clustering. Taken together, the α-Syn-lysoPC interaction may play a role in α-Syn function.

Single-cell multiomics techniques have been widely applied to detect the key signature of cells. These methods have achieved a single-molecule resolution and can even reveal spatial localization. These emerging methods provide insights elucidating the features of genomic, epigenomic and transcriptomic heterogeneity in individual cells. However, they have given rise to new computational challenges in data processing. Here, we describe Single-cell Single-molecule multiple Omics Pipeline (ScSmOP), a universal pipeline for barcode-indexed single-cell single-molecule multiomics data analysis. Essentially, the C language is utilized in ScSmOP to set up spaced-seed hash table-based algorithms for barcode identification according to ligation-based barcoding data and synthesis-based barcoding data, followed by data mapping and deconvolution. We demonstrate high reproducibility of data processing between ScSmOP and published pipelines in comprehensive analyses of single-cell omics data (scRNA-seq, scATAC-seq, scARC-seq), single-molecule chromatin interaction data (ChIA-Drop, SPRITE, RD-SPRITE), single-cell single-molecule chromatin interaction data (scSPRITE) and spatial transcriptomic data from various cell types and species. Additionally, ScSmOP shows more rapid performance and is a versatile, efficient, easy-to-use and robust pipeline for single-cell single-molecule multiomics data analysis.

Measuring molecular binding kinetics represents one of the most important tasks in molecular interaction analysis. Surface plasmon resonance (SPR) is a popular tool for analyzing molecular binding. Plasmonic scattering microscopy (PSM) is a newly developed SPR imaging technology, which detects the out-of-plane scattering of surface plasmons by analytes and has pushed the detection limit of label-free SPR imaging down to a single-protein level. In addition, PSM also allows SPR imaging with high spatiotemporal resolution, making it possible to analyze cellular response to the molecular bindings. In this Mini Review, we present PSM as a method of choice for chemical and biological imaging, introduce its theoretical mechanism, present its experimental schemes, summarize its exciting applications, and discuss its challenges as well as the promising future.

Detecting diseases at the molecular level aids in early diagnosis and treatment. However, traditional immunological detection techniques, such as enzyme-linked immunosorbent assay (ELISA) and chemiluminescence, have detection sensitivities between 10-16 and 10-12 mol/L, which are inadequate for early diagnosis. Single-molecule immunoassays can reach detection sensitivities of 10-18 mol/L and can detect biomarkers that are difficult to measure using conventional detection techniques. It can confine molecules to be detected in a small spatial area and provide absolute counting of the detected signal, offering the advantage of high efficiency and accuracy. Herein, we demonstrate the principles and equipment of two single-molecule immunoassay techniques and discuss their applications. It is shown that the detection sensitivity can be improved by 2-3 orders of magnitude compared to common chemiluminescence or ELISA assays. The microarray-based single-molecule immunoassay technique can test 66 samples in 1 h, which is more efficient than conventional immunological detection techniques. In contrast, microdroplet-based single-molecule immunoassay techniques can generate 107 droplets in 10 min, which is more than 100 times faster than a single droplet generator. By comparing the two single-molecule immunoassay techniques, we highlight our personal perspectives on the current limitations of point-of-care applications and future development trends.

Cell membranes are complicated multicomponent structures, related to many basic cellular processes, such as substance transporting, energy conversion, signal transduction, mechanosensing, cell adhesion and so on. However, cell membranes have long been difficult to study at a single-molecule level due to their complex and dynamic properties. During the last decades, biophysical imaging techniques, such as atomic force microscopy and super-resolution fluorescent microscopy, have been developed to study biological structures with unprecedented resolution, enabling researchers to analyze the composition and distribution of membrane proteins and monitor their specific functions at single cell/molecule level. In this review, we highlight the structure and functions of cell membranes based on up-to-date biophysical techniques. Additionally, we describe the recent advances in force-based detecting technology, which allow insight into dynamic events and quantitativelymonitoring kinetic parameters for trans-membrane transporting in living cells.