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Results for “fluorescence correlation spectroscopy (FCS)”

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12 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Polymer Size–Catalytic Activity Relationships in Solution by Fluorescence Correlation Spectroscopy

Measuring the catalytic activity of specific sizes of polymers with active catalysts in solution is typically challenging, due to limited instrument detection sensitivity and/or dynamic range. Here, a fluorescence correlation spectroscopy (FCS) method is developed to determine the catalytic activity of living polymers of a specific apparent size in solution. Deviation from a single-component FCS data fitting, as assessed by χ2, is also introduced and developed as a “speciation index”—a method to evaluate and track changes in the relative amount of distinct polymer sizes with reaction progress. These methods are enabled by incorporating a selectively reactive fluorescent monomer into growing polydicyclopentadiene or polynorbornene during ring-opening metathesis polymerization (ROMP). Compared to polynorbornene, data showed that catalysts in aggregates of polyDCPD retained higher activity for longer—outcomes not directly inferable from simple diffusional-access predictions. Here, the ability to assign catalytic activity to polymers of specific sizes, and then to determine how this activity evolves with reaction progress, support long-term goals in the development and measurement of nano-objects that possess size-dependent catalytic activity.

Active catalyst↗
DOE OSTI2022

Investigation of Molecular Diffusion at Block Copolymer Thin Films Using Maximum Entropy Method-Based Fluorescence Correlation Spectroscopy and Single Molecule Tracking

Fluorescence correlation spectroscopy (FCS) has been widely used to investigate molecular diffusion behavior in various samples. The use of the maximum entropy method (MEM) for FCS data analysis provides a unique means to determine multiple distinct diffusion coefficients without a priori assumption of their number. Comparison of the MEM-based FCS method (MEM-FCS) with another method will reveal its utility and advantage as an analytical tool to investigate diffusion dynamics. Herein, we measured diffusion of fluorescent probes doped into nanostructured thin films using MEM-FCS, and validated the results with single molecule tracking (SMT) data. The efficacy of the MEM code employed was first demonstrated by analyzing simulated FCS data for systems incorporating one and two diffusion modes with broadly distributed diffusion coefficients. The MEM analysis accurately afforded the number of distinct diffusion modes and their mean diffusion coefficients. These results contrasted with those obtained by fitting the simulated data to conventional two-component and anomalous diffusion models, which yielded inaccurate estimates of the diffusion coefficients. Subsequently, the MEM analysis was applied to FCS data acquired from hydrophilic dye molecules incorporated into microphase-separated polystyrene-block-poly(ethylene oxide) (PS-b-PEO) thin films characterized under a water-saturated nitrogen atmosphere. The MEM analysis revealed distinct fast and slow diffusion components attributable to molecules diffusing on the film surface and inside the film, respectively. SMT studies of the same materials yielded trajectories for mobile molecules that appear to follow the curved PEO microdomains. Diffusion coefficients obtained from the SMT data were consistent with those obtained for the slow diffusion component detected by MEM-FCS. Furthermore, these results highlight the utility of MEM-FCS and SMT for gaining complementary information on molecular diffusion processes in heterogeneous material systems.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Biophysical Characterization of Membrane Proteins Embedded in Nanodiscs Using Fluorescence Correlation Spectroscopy

Proteins embedded in biological membranes perform essential functions in all organisms, serving as receptors, transporters, channels, cell adhesion molecules, and other supporting cellular roles. These membrane proteins comprise ~30% of all human proteins and are the targets of ~60% of FDA-approved drugs, yet their extensive characterization using established biochemical and biophysical methods has continued to be elusive due to challenges associated with the purification of these insoluble proteins. In response, the development of nanodisc techniques, such as nanolipoprotein particles (NLPs) and styrene maleic acid polymers (SMALPs), allowed membrane proteins to be expressed and isolated in solution as part of lipid bilayer rafts with defined, consistent nanometer sizes and compositions, thus enabling solution-based measurements. Fluorescence correlation spectroscopy (FCS) is a relatively simple yet powerful optical microscopy-based technique that yields quantitative biophysical information, such as diffusion kinetics and concentrations, about individual or interacting species in solution. Here, we first summarize current nanodisc techniques and FCS fundamentals. We then provide a focused review of studies that employed FCS in combination with nanodisc technology to investigate a handful of membrane proteins, including bacteriorhodopsin, bacterial division protein ZipA, bacterial membrane insertases SecYEG and YidC, Yersinia pestis type III secretion protein YopB, yeast cell wall stress sensor Wsc1, epidermal growth factor receptor (EGFR), ABC transporters, and several G protein-coupled receptors (GPCRs).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Random Walks and Sticky Surfaces: Single-Molecule Measurements of Solute Diffusion in Ethanol/Water-Filled Anodic Alumina Nanopores

Nanoporous anodic aluminum oxide (AAO) membranes are now being explored for use in advanced chemical separations, including in the dehydration of biofuels such as ethanol. Optimization of membrane performance requires an in-depth understanding of how solvent mixtures and solutes behave under nanoconfinement. In this work, the diffusion of rhodamine B (RhB) dye through 10 nm and 20 nm AAO nanopores filled with a series of ethanol/water mixtures (0 - 33% water) is explored by fluorescence correlation spectroscopy (FCS). RhB was found to diffuse through the pores by two distinct mechanisms with mean diffusion coefficients, $D_f$ and $D_s$, reflecting fast and slow diffusive motions, respectively, with values that differ by nearly two orders of magnitude. Further, both $D_f$ and $D_s$ increased with pore size and were significantly smaller than $D_b$, the RhB diffusion coefficient in bulk liquid. Mean $D_f$ values follow a composition-dependent trend that closely mimics the viscosity dependence of $D_b$. Additional slowing of fast RhB diffusion is attributed to both hydrodynamic drag and electrostatic interactions with the nanopore surface. The mean $D_s$ values exhibit a different trend with increasing water content, revealing an increase in $D_s$ and a decrease in the contributions of slow diffusion to the observed dynamics. The fluorescence time transient data used in the analysis show that the slow diffusion process is strongly hindered and likely involves frequent adsorption of RhB to the pore surfaces. These results provide new insights into the detailed molecular-level mechanisms of mass transport in nanoporous AAO membranes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Immobilization of Cyanines in DNA Produces Systematic Increases in Fluorescence Intensity

Cyanines are useful fluorophores for a myriad of biological labeling applications, but their interactions with biomolecules are unpredictable. Cyanine fluorescence intensity can be highly variable due to complex photoisomerization kinetics, which are exceedingly sensitive to the surrounding environment. This introduces large errors in Förster resonance energy transfer (FRET)-based experiments where fluorescence intensity is the output parameter. However, this environmental sensitivity is a strength from a biological sensing point of view if specific relationships between biomolecular structure and cyanine photophysics can be identified. We describe a set of DNA structures that modulate cyanine fluorescence intensity through the insertion of adenine or thymine bases. These structures simultaneously provide photophysical predictability and tunability. We characterize these structures using steady-state fluorescence measurements, fluorescence correlation spectroscopy (FCS), and time-resolved photoluminescence (TRPL). Here, we find that the photoisomerization rate decreases over an order of magnitude across the adenine series, which is consistent with increasing immobilization of the cyanine moiety by the surrounding DNA structure.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Spectroscopic and Chemical Properties of Ionic Liquids: Computational Study

A brief account is given of highlights of our computational efforts – often in collaboration with experimental groups – to understand spectroscopic and chemical properties of ionic liquids (ILs). Molecular dynamics, including their inhomogeneous character, responsible for key spectral features observed in dielectric absorption, infra-red (IR) and fluorescence correlation spectroscopy (FCS) measurements are elucidated. Mechanisms of chemical processes involving imidazolium-based ILs are illustrated for CO 2 capture and related reactions, transesterification of cellulose, and Au nanocluster-catalyzed Suzuki cross-coupling reaction with attention paid to differing roles of IL ions. A comparison with experiments is also made.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Ligand modulation of the conformational dynamics of the A 2A adenosine receptor revealed by single-molecule fluorescence

G protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins, making them an important target for therapeutics. Activation of these receptors is modulated by orthosteric ligands, which stabilize one or several states within a complex conformational ensemble. The intra- and inter-state dynamics, however, is not well documented. Here, we used single-molecule fluorescence to measure ligand-modulated conformational dynamics of the adenosine A 2A receptor (A 2A R) on nanosecond to millisecond timescales. Experiments were performed on detergent-purified A 2R in either the ligand-free (apo) state, or when bound to an inverse, partial or full agonist ligand. Single-molecule Förster resonance energy transfer (smFRET) was performed on detergent-solubilized A 2A R to resolve active and inactive states via the separation between transmembrane (TM) helices 4 and 6. The ligand-dependent changes of the smFRET distributions are consistent with conformational selection and with inter-state exchange lifetimes ≥ 3 ms. Local conformational dynamics around residue 229 6.31 on TM6 was measured using fluorescence correlation spectroscopy (FCS), which captures dynamic quenching due to photoinduced electron transfer (PET) between a covalently-attached dye and proximal aromatic residues. Global analysis of PET-FCS data revealed fast (150–350 ns), intermediate (50–60 μs) and slow (200–300 μs) conformational dynamics in A 2A R, with lifetimes and amplitudes modulated by ligands and a G-protein mimetic (mini-G s ). Most notably, the agonist binding and the coupling to mini-G s accelerates and increases the relative contribution of the sub-microsecond phase. Molecular dynamics simulations identified three tyrosine residues (Y112, Y288 7.53 , and Y290 7.55 ) as being responsible for the dynamic quenching observed by PET-FCS and revealed associated helical motions around residue 229 6.31 on TM6. As a result, this study provides a quantitative description of conformational dynamics in A 2A R and supports the idea that ligands bias not only GPCR conformations but also the dynamics within and between distinct conformational states of the receptor.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2002

Two-Photon Fluorescence Correlation Spectroscopy

We will describe a two-photon microscope currently under development at the NASA Glenn Research Center. It is composed of a Coherent Mira 900 tunable, pulsed Titanium:Sapphire laser system, an Olympus Fluoview 300 confocal scanning head, and a Leica DM IRE inverted microscope. It will be used in conjunction with a technique known as fluorescence correlation spectroscopy (FCS) to study intracellular protein dynamics. We will briefly explain the advantages of the two-photon system over a conventional confocal microscope, and provide some preliminary experimental results.

Zimmerli, Gregory A.↗
DOE OSTI2020

Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments

Significance Recent experiments have suggested that the energy released by a chemical reaction can propel its enzyme catalyst (for example, alkaline phosphatase). However, this topic remains controversial, partially due to the indirect and ensemble nature of existing measurements. Here, we used recently developed single-molecule approaches to monitor directly the motions of individual proteins in aqueous solution and find that single alkaline phosphatase enzymes do not diffuse faster under catalysis. Instead, we demonstrate that interactions between the fluorescent dye and the enzyme’s substrate can produce the signature of apparent diffusion enhancement in fluorescence correlation spectroscopy, the standard ensemble assay currently used to study enzyme diffusion and indicate that single-molecule approaches provide a more robust means to investigate diffusion at the nanoscale.

36 MATERIALS SCIENCE↗
DOE OSTI2020

Influences of Hydrogen Bonding-Based Stabilization of Bolaamphiphile Layers on Molecular Diffusion within Organic Nanotubes Having Inner Carboxyl Groups

This paper reports molecular diffusion behavior in two bolaamphiphile-based organic nanotubes having inner carboxyl groups with different inner dimeters (10 and 20 nm) and wall structures, COOH-ONT 10nm and COOH-ONT 20nm , using imaging fluorescence correlation spectroscopy (imaging FCS). Here, the results were compared to those previously obtained in a similar nanotube with inner amine groups (NH 2 -ONT 10nm ). COOH-ONT 10nm , as with NH 2 -ONT 10nm , were formed from a rolled bolaamphiphile layer incorporating triglycine moieties, whereas COOH-ONT 20nm consisted of four stacks of triglycine-free bolaamphiphile layers. Imaging FCS measurements were carried out for anionic sulforhodamine B (SRB), zwitterionic/cationic rhodamine B (RB) and cationic rhodamine-123 (R123) diffusing within ONTs (1–9 µm long) at different pH (3.4–8.4) and ionic strengths (1.6–500 mM). Diffusion coefficients (D) of these dyes in the ONTs were very small (0.01–0.1 µm 2 /s), reflecting the significant contributions of molecule-nanotube interactions to diffusion. The D of SRB was larger at higher pH and ionic strength, indicating the essential role of electrostatic repulsion that was enhanced by the deprotonation of the inner carboxyl groups. Importantly, the D of SRB was virtually independent of nanotube inner diameter and wall structure, indicating the diffusion of the hydrophilic molecule was controlled by short timescale adsorption/desorption processes onto the inner surface. In contrast, pH effects on D were less clear for relatively hydrophobic R123 and RB, suggesting the significant contributions of non-Coulombic interactions. Interestingly, the diffusion of these molecules in COOH-ONT 20nm was slower than in COOH-ONT 10nm . Slower diffusion in COOH-ONT20nm was attributable to relatively efficient partitioning of the hydrophobic dyes into the bolaamphiphile layers, which was reduced in COOH-ONT 10nm due to the stabilization of its layer by polyglycine-II-type hydrogen bonding networks. These results show that, by tuning the bolaamphiphile structures and their intermolecular interactions, unique environments can be created within the nanospaces for enhanced molecular separations and reactions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Diffusion of Hydrophilic to Hydrophobic Forms of Nile Red in Aqueous C 12 EO 10 Gels by Variable Area Fluorescence Correlation Spectroscopy

In this work, solute diffusion within lyotropic liquid crystal gels prepared from a series of water and decaethylene glycol monododecyl ether (C 12 EO 10 ) mixtures was explored by variable area fluorescence correlation spectroscopy. Aqueous C 12 EO 10 gels were prepared in concentrations ranging from 55:45 to 70:30 wt% of surfactant and water. Small angle X-ray scattering revealed that these gels comprise hexagonal mesophases of cylindrical micelles. Micelle spacing was found to decrease with increasing C 12 EO 10 concentration. Three different Nile red (NR) dyes were employed as model solutes and were separately doped into the gels at nanomolar levels. These include a hydrophilic form of NR incorporating an anionic sulfonate group (NRSO 3 - ), a hydrophobic form incorporating a fourteen-carbon alkane tail (NRC 14 ), and commercial NR as an intermediate case. FCS data acquired from the gels revealed that NRSO 3 - diffused primarily in 3D. Its diffusion coefficient exhibited a monotonic decrease with increasing gel concentration and micelle packing density, consistent with confinement of its motions as a result of its exclusion from the micelle cores. NRC 14 exhibited the smallest diffusion coefficient, most likely due to its larger size and enhanced interactions with the micelle cores. NR yielded an intermediate diffusion coefficient and the most anomalous behavior of the three dyes, attributable to its facile partitioning between core and corona regions, and greater participation by 1D diffusion. The results of these studies afford an improved understanding of molecular mass transport through soft-matter nanomaterials like those being developed for use in drug delivery and membrane based chemical separations.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Overcoming Barriers Associated with Oral Delivery of Differently Sized Fluorescent Core‐Shell Silica Nanoparticles

Oral delivery, while a highly desirable form of nanoparticle-drug administration, is limited by challenges associated with overcoming several biological barriers. Here, in this study, the authors study how fluorescent and poly(ethylene glycol)-coated (PEGylated) core-shell silica nanoparticles sized 5 to 50 nm interact with major barriers including intestinal mucus, intestinal epithelium, and stomach acid. From imaging fluorescence correlation spectroscopy studies using quasi-total internal reflection fluorescence microscopy, diffusion of nanoparticles through highly scattering mucus is progressively hindered above a critical hydrodynamic size around 20 nm. By studying Caco-2 cell monolayers mimicking the intestinal epithelia, it is observed that ultrasmall nanoparticles below 10 nm diameter (Cornell prime dots, [C’ dots]) show permeabilities correlated with high absorption in humans from primarily enhanced passive passage through tight junctions. Particles above 20 nm diameter exclusively show active transport through cells. After establishing C’ dot stability in artificial gastric juice, in vivo oral gavage experiments in mice demonstrate successful passage through the body followed by renal clearance without protein corona formation. Results suggest C’ dots as viable candidates for oral administration to patients with a proven pathway towards clinical translation and may generate renewed interest in examining silica as a food additive and its effects on nutrition and health.

36 MATERIALS SCIENCE↗