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[Advances in Experimental Medicine and Biology] Glycobiophysics Volume 1104 || Unraveling of Lipid Raft Organization in Cell Plasma Membranes by Single-Molecule Imaging of Ganglioside Probes
摘要： Gangliosides are involved in a variety of physiological roles and particularly in the formation and function of lipid rafts in cell membranes. However, the dynamic behaviors of gangliosides have not been investigated in living cells owing to the lack of fluorescent probes that behave like their parental molecules. This has recently been resolved by developing new fluorescent ganglioside analogues that act similarly to their parental molecules, synthesized by only chemical methods. We performed single fluorescent-molecule imaging and revealed that ganglioside probes dynamically enter and exit rafts containing CD59, a glycosylphosphatidylinositol (GPI)-anchored protein, both before and after stimulation. The residency time of our ganglioside probes in CD59 oligomers was 48 ms after stimulation. The residency times in CD59 homodimer and monomer rafts were 40 and 12 ms, respectively. These results reveal the first direct evidence that GPI-anchored receptors and gangliosides interact in a cholesterol-dependent manner. Furthermore, they demonstrate that gangliosides continually move in and out of rafts that contain CD59 in an extremely dynamic manner and at a much higher frequency than expected. In this chapter, we review methods for the development and single-molecule imaging of new fluorescent ganglioside analogues and discuss how raft domains are formed, both before and after receptor engagement.
关键词： GPI-anchored protein,Single-molecule fluorescence tracking,Cell plasma membrane,Raft domains,Fluorescent ganglioside probes
更新于2025-09-10 09:29:36
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Absorption and Raman scattering by molecule adsorbed on crystal surface: many particles approach
摘要： The spectroscopy of an organic molecule adsorbed on the surface of a crystal is studied using the many particle approach. The interaction between electrons and vibrations of the molecule as well as the interaction of crystal electrons between each other and with the electrons of the molecule is taken into account. It is supposed that molecules adsorbed on the surface do not interact one with another directly but rather such interaction arises by the electrons of the crystal. All these interactions give rise to a very complicated dependence of the spectrum. Because all molecules are arbitrarily distributed on the crystal surface the symmetry of the system molecule-crystal surface is small, C1, and so all forbidden rules for absorption and Raman scattering (RS) processes are identical. Numerical simulation of the frequency dependence of absorption (or RS) coefficient, obtained in the framework of micro-approach using the Green function method, showed that at some relations between energy excitation of the molecule and the gap between the electron bands of the crystal film, and also the position of the Fermi level, can result in significant enhancement of absorption (or RS), relative to the free molecule, in the complex molecule-crystal film.
关键词： many particles approach,molecule,crystal surface,Raman scattering,Absorption
更新于2025-09-10 09:29:36
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Kinetics of Transient Protein Complexes Determined via Diffusion-Independent Microfluidic Mixing and Fluorescence Stoichiometry
摘要： Low-a?nity protein complexes and their transient states are di?cult to measure in single-molecule experiments because of their low concentrations. A prominent solution to this problem is the use of micro?uidic mixing devices, which rely on di?usion-based mixing. This is not ideal for multiprotein complexes, as the single-molecule ?uorescence signal is dominated by the already dissociated species. Here, we designed a micro?uidic device with mixing structures for fast and homogeneous mixing of components with varying di?usion coe?cients and for ?uorescence measurements at a de?ned single-molecule concentration. This enables direct measurement of dissociation rates at a broad range of timescales from a few milliseconds to several minutes. This further allows us to measure structural properties and stoichiometries of protein complexes with large equilibrium dissociation constants (KD’s) of 5 μM and above. We used the platform to measure structural properties and dissociation rates of heat shock protein 90 (Hsp90) dimers and found at least two dissociation rates which depend on the nucleotide state. Finally, we demonstrate the capability for measuring also equilibrium dissociation constants, resulting in the determination of both the kinetics and thermodynamics of the system under investigation.
关键词： single-molecule ?uorescence,micro?uidic mixing,dissociation rates,Hsp90,protein complexes
更新于2025-09-10 09:29:36
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Two Highly Stable Luminescent Lead Phosphonates Based on Mixed Ligands: Highly Selective and Sensitive Sensing for Thymine Molecule and VO <sub/>3</sub><sup>–</sup> Anion
摘要： Two luminescent lead phosphonates with two-dimensional (2D) layer and three-dimensional (3D) framework structure, namely, Pb3[(L1)2(Hssc)(H2O)2] (1) and [Pb2(L2)0.5(bts)(H2O)2]·H2O (2) (H2L1 = O(CH2CH2)2NCH2PO3H2, H4L2 = H2PO3CH2NH(C2H4)2NHCH2PO3H2, H3ssc = 5-sulfosalicylic acid, NaH2bts = 5-sulfoisophthalic acid sodium) have been prepared via hydrothermal techniques. The two compounds not only show excellent thermal stability but also remain intact in aqueous solution within an extensive pH range. Moreover, the atomic absorption spectroscopy analysis experiment indicates that there does not exist the leaching of Pb2+ ions from the lead phosphonates, which show they are nontoxic in aqueous solution. In compound 1, the Pb(1)O4, Pb(2)O7, Pb(3)O4, and CPO3 polyhedra are interlinked into a one-dimensional chain, which is further connected to adjacent chain by sharing the Hssc2? to form a 2D layer. Interestingly, compound 1 as a highly selective and sensitive luminescent material can be used to detect the thymine molecule with a very low detection limit of 8.26 × 10?7 M. In compound 2, the Pb(1)O6 and Pb(2)O5 polyhedra are interlinked into a dimer via edge sharing, which is further connected to adjacent dimer to form a tetramer via corner sharing, and such a tetramer is then interlinked into a 2D layer through bts3? ligands; the adjacent 2D layers are finally constructed to a 3D structure by sharing the L24? ligand. Compound 2 can be applied as an excellent luminescent sensor for sensing of VO3? anion. Furthermore, the probable fluorescent quenching mechanisms of the two compounds have also been studied.
关键词： chemical stability,luminescent lead phosphonates,VO3? anion,thermal stability,hydrothermal techniques,fluorescent quenching mechanisms,thymine molecule
更新于2025-09-10 09:29:36
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Super Blinking and Biocompatible Nanoprobes Based on Dye Doped BSA Nanoparticles for Super Resolution Imaging
摘要： As one of the super-resolved optical imaging technique, single molecule localization microscopy (SMLM) imaging techniques, SMLM has one particular request for the fluorophores, that is, continuous “on” and “off” behaviors of their signals (referred to as “blinking”). Hence, we present here a kind of Super Blinking and biocompatible Nanoprobes (denoted as SBNs) for SMLM. The SBNs have two main advantages, first, they possess an outstanding fluorescence blinking. Second, they are biocompatible since they are based on bovine serum albumin (BSA). The SBNs are fabricated by doping organic dyes into BSA nanoparticles (NPs). We fabricated two kinds of SBNs, one was doped with Alexa Fluor 647 (A647) and the other was doped with Alexa Fluor 594 (A594). Especially for A594 doped SBNs, the improved blinking of A594 doped SBNs induced a better localization precision as compared with A594 alone. Moreover, SMLM imaging of breast cancer cells and exosomes using the SBNs was successfully realized with high spatial resolutions. The work demonstrated here provides a new strategy to prepare novel kinds of super blinking fluorescent agents for SMLM, which broadens the selection of suitable fluorophores for SMLM.
关键词： BSA nanoparticles,single molecule localization microscopy,super blinking,dyes,localization precision
更新于2025-09-10 09:29:36
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An approach to spin-resolved molecular gas microscopy
摘要： Ultracold polar molecules are an ideal platform for studying many-body physics with long-range dipolar interactions. Experiments in this field have progressed enormously, and several groups are pursuing advanced apparatus for manipulation of molecules with electric fields as well as single-atom-resolved in situ detection. Such detection has become ubiquitous for atoms in optical lattices and tweezer arrays, but has yet to be demonstrated for ultracold polar molecules. Here we present a proposal for the implementation of site-resolved microscopy for polar molecules, and specifically discuss a technique for spin-resolved molecular detection. We use numerical simulation of spin dynamics of lattice-confined polar molecules to show how such a scheme would be of utility in a spin-diffusion experiment.
关键词： quantum gas microscopy,quantum simulation,ultracold molecules,single-molecule control,dipolar spin models,optical lattices
更新于2025-09-09 09:28:46
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Selective negative-ion formation from core-valence doubly excited states of the water molecule
摘要： This study focuses on the role of negative-ion formation in the decay of core-valence doubly excited water molecules. Combining negative- and positive-ion coincidence measurements with calculated energies of core-valence doubly excited states, we find that O? and H? production is enhanced selectively. In particular, we suggest that O? production is correlated to double occupancy of the antibonding 4a1 virtual orbital, while H? appears at electronic states with double occupancy in 2b2. We also show that H? and O? can be created as a result of electron recapture close to the O 1s ionization potential.
关键词： core-valence doubly excited states,electron recapture,negative-ion formation,water molecule,O 1s ionization potential
更新于2025-09-09 09:28:46
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Characterization of functionalized glass and indium tin oxide surfaces as substrates for super-resolution microscopy
摘要： Modern high-throughput biosensors with sensitivity down to a single analyte molecule may be possible with single-molecule localization microscopy (SMLM). Functionalized surfaces can be fabricated with self-assembly monolayer chemistry on indium tin oxide (ITO) substrates but not glass. However, characterizations of SMLM-compatible fluorophores are primarily performed on glass substrates. Here we collect single-molecule kinetics data of isolated Alexa Fluor 647 molecules on bare and functionalized glass and ITO surfaces. Extracting the photophysical dynamics of the fluorophores allows direct comparison of behavior of this dye on these substrates and fitting data to a model that accounts for multiple reversible dark states. All surfaces had sensitivity sufficient to image single fluorophore molecules. Photophysical kinetics observed are similar between the two substrates. The photon yield from individual fluorophores was greatest on bare glass, but functionalized ITO surfaces showed superior yield to functionalized glass surfaces and nearly matched the yield of bare glass. Together these results indicate functionalized ITO as a promising substrate for modern single-molecule biosensors.
关键词： indium tin oxide,super-resolution microscopy,biosensor,single-molecule imaging
更新于2025-09-09 09:28:46
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[Methods in Molecular Biology] Atomic Force Microscopy Volume 1886 (Methods and Protocols) || Molecular Recognition Force Spectroscopy for Probing Cell Targeted Nanoparticles In Vitro
摘要： In the development and design of cell targeted nanoparticle-based systems the density of targeting moieties plays a fundamental role in allowing maximal cell-specific interaction. Here, we describe the use of molecular recognition force spectroscopy as a valuable tool for the characterization and optimization of targeted nanoparticles toward attaining cell-specific interaction. By tailoring the density of targeting moieties at the nanoparticle surface, one can correlate the unbinding event probability between nanoparticles tethered to an atomic force microscopy tip and cells to the nanoparticle vectoring capacity. This novel approach allows for a rapid and cost-effective design of targeted nanomedicines reducing the need for long and tedious in vitro tests.
关键词： Targeted nanoparticles,Tailored nanomedicine,Tip functionalization,Single molecule force spectroscopy,Drug delivery
更新于2025-09-09 09:28:46
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Synthesis of Novel Donor-Acceptor Type Molecule Based on Phenothiazine Unit for Organic Light-Emitting Materials
摘要： A novel conjugated organic molecule based on central phenothiazine unit as an electronic donor and aldehyde group termini as an electronic acceptor has been designed and constructed. Optical spectra show that, the absorption and fluorescence spectra of M2 are large red-shifted relative to M1 in both states, however, from solution to the solid state, the absorption maxima and emission maxima of M1 and M2 are in the opposite trends (blue-shift). The fluorescence spectra exhibit that the emission maxima of M1 and M2 which range from 485 nm to 588 nm belong to green light region. As expected, the target compounds should be a promising green light-emitting material for applications in organic light-emitting diodes (OLEDs).
关键词： Molecule,Donor-acceptor,Phenothiazine unit,Light-emitting materials
更新于2025-09-09 09:28:46