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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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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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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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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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Real-time parallel 3D multiple particle tracking with single molecule centrifugal force microscopy
摘要： Real-time tracking of multiple particles is key for quantitative analysis of dynamic biophysical processes and materials science via time-lapse microscopy image data, especially for single molecule biophysical techniques, such as magnetic tweezers and centrifugal force microscopy. However, real-time multiple particle tracking with high resolution is limited by the current imaging processes or tracking algorithms. Here, we demonstrate 1 nm resolution in three dimensions in real-time with a graphics-processing unit (GPU) based on a compute unified device architecture (CUDA) parallel computing framework instead of only a central processing unit (CPU). We also explore the trade-offs between processing speed and size of the utilized regions of interest and a maximum speedup of 137 is achieved with the GPU compared with the CPU. Moreover, we utilize this method with our recently self-built centrifugal force microscope (CFM) in experiments that track multiple DNA-tethered particles. Our approach paves the way for high-throughput single molecule techniques with high resolution and efficiency.
关键词： image processing,single molecule force spectroscopy,GPU,particle tracking
更新于2025-09-09 09:28:46
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Sharpening emitter localization in front of a tuned mirror
摘要： Single-molecule localization microscopy (SMLM) aims for maximized precision and a high signal-to-noise ratio. Both features can be provided by placing the emitter in front of a metal-dielectric nanocoating that acts as a tuned mirror. Here, we demonstrate that a higher photon yield at a lower background on biocompatible metal-dielectric nanocoatings substantially improves SMLM performance and increases the localization precision by up to a factor of two. The resolution improvement relies solely on easy-to-fabricate nanocoatings on standard glass coverslips and is spectrally and spatially tunable by the layer design and wavelength, as experimentally demonstrated for dual-color SMLM in cells.
关键词： localization precision,metal-dielectric nanocoatings,dual-color SMLM,Single-molecule localization microscopy
更新于2025-09-09 09:28:46
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Diffracted X-ray Blinking Tracks Single Protein Motions
摘要： Single molecule dynamics studies have begun to use quantum probes. Single particle analysis using cryo-transmission electron microscopy has dramatically improved the resolution when studying protein structures and is shifting towards molecular motion observations. X-ray free-electron lasers are also being explored as routes for determining single molecule structures of biological entities. Here, we propose a new X-ray single molecule technology that allows observation of molecular internal motion over long time scales, ranging from milliseconds up to 103 seconds. Our method uses both low-dose monochromatic X-rays and nanocrystal labelling technology. During monochromatic X-ray diffraction experiments, the intensity of X-ray diffraction from moving single nanocrystals appears to blink because of Brownian motion in aqueous solutions. X-ray diffraction spots from moving nanocrystals were observed to cycle in and out of the Bragg condition. Consequently, the internal motions of a protein molecule labelled with nanocrystals could be extracted from the time trajectory using this diffracted X-ray blinking (DXB) approach. Finally, we succeeded in distinguishing the degree of fluctuation motions of an individual acetylcholine-binding protein (AChBP) interacting with acetylcholine (ACh) using a laboratory X-ray source.
关键词： Acetylcholine-binding protein,Brownian motion,Nanocrystal labelling,Single molecule dynamics,X-ray diffraction
更新于2025-09-09 09:28:46
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Comparative Study of Single Molecular Junctions with <i>Para</i> -Phthalic Acid and <i>Meta</i> -Phthalic Acid Binding to Different Metal Electrodes
摘要： In this paper, single molecular junctions of para-phthalic acid and meta-phthalic acid with Au electrodes were studied by STM break junction approach. Conductance values of 10?3(cid:2)55 G0 and 10?3(cid:2)70 G0 were found for para-phthalic acid and meta-phthalic acid, respectively. The conductance order between para-phthalic acid and meta-phthalic acid with Au is different from that with Cu, which can be contributed to the different coupling between molecules and electrodes; different through-space interaction is proposed for such phenomenon between Cu and Au electrodes. Furthermore, the breaking off distances can re?ect the length of molecules. The current work presents the important role of electrode in single molecular junctions with different position anchoring groups.
关键词： STM-BJ,Meta-Phthalic Acid,Carboxylic Acid,Para-Phthalic Acid,Single-Molecule Junctions
更新于2025-09-04 15:30:14