Single molecule microscopy

With recent advances in DNA-templated dye aggregation for leveraging and engineering molecular excitons, a need exists for minimizing structural heterogeneity. Holliday Junction complexes (HJ) are commonly used to covalently template dye aggregates on their core; however, the global conformation of HJ is detrimentally dynamic. Here, the global conformation of the HJ is selectively tuned by restricting its position and orientation by using a sheet-like DNA origami construct (DOC) physisorbed on glass. The HJ arms are fixed with four different designed interduplex angles (IDAs)...

Using scanning tunneling microscopy (STM), we experimentally and theoretically investigate isolated platinum phthalocyanine (PtPc) molecules adsorbed on an atomically thin NaCl(100) film vapor deposited on Au(111). We obtain good agreement between theory and constant-height STM topography. We theoretically examine why strong distortions of STM images occur as a function of distance between the molecule and the STM tip. The images of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) exhibit for increasing distance, significant radial expansion due to electron propagation in the vacuum...

Single-molecule multiplexed detection is a high-promise toolkit for the expanding field of biosensing and molecular diagnostics. Among many single-molecule techniques available today for biomarker sensing including fluorescence, force, electrochemical, spectroscopic, barcoding, and other techniques, fluorescence-based approaches are arguably the most widely used methods due to their high sensitivity, selectivity, and readily available fluorophore-labeling schemes for a wide variety of biomolecules. However, multiplexed imaging using fluorescence techniques has proven to be challenging due to the sophisticated labeling schemes often requiring multiple FRET (fluorescence resonance energy transfer) pairs and/or excitation sources, which lead to overlapping signals and complicate data analysis...

Single-molecule localization microscopy (SMLM), a type of super-resolution fluorescence microscopy, has become a strong technique in the toolbox of chemists, biologists, physicists, and engineers in recent years for its unique ability to resolve characteristic features at the nanoscopic level. It drastically improves the resolution of optical microscopes beyond the diffraction limit, with which previously unresolvable structures can now be studied. Spectrally resolved super-resolution fluorescence microscopy via multiplexing of different fluorophores is one of the greatest advancements among SMLM techniques...

Aquaporin-4 (AQP4) facilitates water transport across astrocytic membranes in the brain, forming highly structured nanometric arrays. AQP4 has a central role in regulating cerebrospinal fluid (CSF) circulation and facilitating the clearance of solutes from the extracellular space of the brain. Adrenergic signaling has been shown to modulate the volume of the extracellular space of the brain via AQP4 localized at the end-feet of astrocytes, but the mechanisms by which AQP4 regulates CSF inflow and outflow in the brain remain elusive...

Core protein components of the abscisic acid (ABA) signaling network, pyrabactin resistance (PYR), protein phosphatases 2C (PP2C), and SNF1-related protein kinase 2 (SnRK2) are involved in the regulation of stomatal closure and gene expression downstream responses in Arabidopsis thaliana . Phosphatidic acid (PA) produced by the phospholipases Dα1 and Dδ (PLDs) in the plasma membrane has been identified as a necessary molecule in ABA-inducible stomatal closure. On the other hand, the involvement of PA in ABA-inducible gene expression has been suggested but remains a question...

Fluorescence imaging is an invaluable tool to study biological processes, and fluorogenic dyes are crucial to enhance cell permeability and target intracellular structures with high specificity. Polymethine dyes are vitally important fluorophores in single-molecule localization microscopy and in vivo imaging, but their use in live cells has been limited by high background fluorescence and low membrane permeability. Here, we present a general strategy to transform polymethine compounds into fluorogenic dyes by implementing a 5-exo-trig ring-closure...

Single-chain polymeric nanoparticles (SCPNs) have been extensively explored as a synthetic alternative to enzymes for catalytic applications. However, the inherent structural heterogeneity of SCPNs, arising from the dispersity of the polymer backbone and stochastic incorporation of different monomers as well as catalytic moieties, is expected to lead to variations in catalytic activity between individual particles. To understand the effect of structural heterogeneities on the catalytic performance of SCPNs, techniques are required that permit researchers to directly monitor SCPN activity at the single-polymer level...

The use of surfactants in oil recovery can effectively improve crude oil recovery rate. Due to the enhanced salt and temperature resistance of surfactant molecules by non-ionic chain segments, anionic groups have good emulsifying stability. Currently, there are many studies on anionic non-ionic surfactants for oil recovery in China, but there is relatively little systematic research on introducing EOs into hydrophobic alkyl chains, especially on their self-assembly behavior. This article proposes a simple and effective synthesis method, using 3-aminopropane sulfonic acid, fatty alcohol polyoxyethylene ether, and epichlorohydrin as raw materials, to insert EO into hydrophobic alkyl chains and synthesize a series of new anionic non-ionic Gemini surfactants (CnEO-5, n = 8, 12, 16)...

Metal-mediated base pairing of DNA has been a topic of extensive research spanning over more than four decades. Precise positioning of a single metal ion by predetermining the DNA sequence, as well as improved conductivity offered by the ions, make these structures interesting candidates in the context of using DNA in nanotechnology. Here, we report the formation and characterization of conjugates of long (kilo bases) homoguanine DNA strands with silver ions. We demonstrate using atomic force microscopy (AFM) and scanning tunneling microscope (STM) that binding of silver ions leads to folding of homoguanine DNA strands in a "hairpin" fashion to yield double-helical, left-handed molecules composed of G-G base pairs each stabilized by a silver ion...

A versatile and robust procedure is developed that allows the identification of individual target molecules using antibodies bound to a DeepTipTM functionalized atomic-force microscopy probe. The model system used for the validation of this process consists of a biotinylated anti-lactate dehydrogenase antibody immobilized on a streptavidin-decorated AFM probe. Lactate dehydrogenase (LDH) is employed as target molecule and covalently immobilized on functionalized MicroDeckTM substrates. The interaction between sensor and target molecules is explored by recording force-displacement (F-z) curves with an atomic-force microscope...

Solid-state nanopores have become a prominent tool in the field of single-molecule detection. Conventional solid-state nanopores are thick, which affects the spatial resolution of the detection results. Graphene is the thinnest 2D material and has the highest spatial detection resolution. In this study, a graphene membrane chip was fabricated by combining a MEMS process with a 2D material wet transfer process. Raman spectroscopy was used to assess the quality of graphene after the transfer. The mechanism behind the influence of the processing dose and residence time of the helium ion beam on the processed pore size was investigated...

Protein@metal-organic frameworks (P@MOFs) prepared by coprecipitation of protein, metal ions, and organic ligands represent an effective method for protein stabilization with a wide spectrum of applications. However, the formation mechanism of P@MOFs via the coprecipitation process and the reason why proteins can retain their biological activity in the frameworks with highly concentrated metal ions remain unsettled. Here, by a combined methodology of single molecule localization microscopy and clustering analysis, we discovered that in this process enzyme molecules form clusters with metal ions and organic ligands, contributing to both the nucleation and subsequent crystal growth...

Since its introduction in 2017, MINFLUX nanoscopy has shown that it can visualise fluorescent molecules with an exceptional localisation precision of a few nanometres. In this overview, we provide a brief insight into technical implementations, fluorescent marker developments and biological studies that have been conducted in connection with MINFLUX imaging and tracking. We also formulate ideas on how MINFLUX nanoscopy and derived technologies could influence bioimaging in the future. This insight is intended as a general starting point for an audience looking for a brief overview of MINFLUX nanoscopy from theory to application...

On-surface synthesis is a powerful method that has emerged recently to fabricate a large variety of atomically precise nanomaterials on surfaces based on polymerization. It is very successful for thermally activated reactions within the framework of heterogeneous catalysis. As a result, it often lacks selectivity. We propose to use selective activation of specific bonds as a crucial ingredient to synthesize desired molecules with high selectivity. In this approach, thermally nonaccessible products are expected to arise in photolytically activated on-surface reactions with high selectivity...

The aim of this work is to shed light on the polymorphism of xanthones, a class of oxygenated molecules well known for their bioactivity, including antioxidant, anticancer, and anti-inflammatory effects. Understanding the polymorphism of xanthones can enable the design of novel solid products for pharmaceutical, nutraceutical, and agrochemical applications. Prior to this work, two entries accounting for different space groups were deposited for 9-xanthone in the Cambridge Structure Database (CSD): an orthorhombic P 21 21 21 and a monoclinic P 21 structure solved at room and low temperatures, respectively...

Microtubules have spatiotemporally complex posttranslational modification patterns. Tubulin tyrosine ligase-like (TTLL) enzymes introduce the most prevalent modifications on α-tubulin and β-tubulin. How TTLLs specialize for specific substrate recognition and ultimately modification-pattern generation is largely unknown. TTLL6, a glutamylase implicated in ciliopathies, preferentially modifies tubulin α-tails in microtubules. Cryo-electron microscopy, kinetic analysis and single-molecule biochemistry reveal an unprecedented quadrivalent recognition that ensures simultaneous readout of microtubule geometry and posttranslational modification status...

State-of-the-art super-resolution microscopy allows researchers to spatially resolve single proteins in dense clusters. However, accurate quantification of protein organization and stoichiometries requires a general method to evaluate absolute binder labeling efficiency, which is currently unavailable. Here we introduce a universally applicable approach that uses a reference tag fused to a target protein of interest. By attaching high-affinity binders, such as antibodies or nanobodies, to both the reference tag and the target protein, and then employing DNA-barcoded sequential super-resolution imaging, we can correlate the location of the reference tag with the target molecule binder...

The endoplasmic reticulum (ER) is a single-copy organelle that cannot be generated de novo, suggesting coordination between the mechanisms overseeing ER integrity and those controlling the cell cycle to maintain organelle inheritance. The Unfolded Protein Response (UPR) is a conserved signaling network that regulates ER homeostasis. Here, we show that pharmacological and genetic inhibition of the UPR sensors IRE1, ATF6, and PERK in unstressed cells delays the cell cycle, with PERK inhibition showing the most penetrant effect, which was associated with a slowdown of the G1 -to-S/G2 transition...

Atomic force microscopy (AFM) has broken boundaries in the characterization of the supramolecular architecture of cell wall assemblies and single cell wall polysaccharides at the nanoscale level. Moreover, AFM provides an opportunity to evaluate the mechanical properties of cell wall material which is not possible with any other method. However, in the case of plant tissue, the critical step is a smart sample preparation that should not affect the polysaccharide structure or assembly and on the other hand should consider device limitations, especially scanner ranges...