Measurement Of Molecular Diffusion Research Articles

Measuring Molecular Diffusion in Dynamic Subcellular Nanostructures by Fast Raster Image Correlation Spectroscopy and 3D Orbital Tracking.

Here we provide demonstration that fast fluorescence fluctuation spectroscopy is a fast and robust approach to extract information on the dynamics of molecules enclosed within subcellular nanostructures (e.g., organelles or vesicles) which are also moving in the complex cellular environment. In more detail, Raster Image Correlation Spectroscopy (RICS) performed at fast timescales (i.e., microseconds) reveals the fast motion of fluorescently labeled molecules within two exemplary dynamic subcellular nanostructures of biomedical interest, the lysosome and the insulin secretory granule (ISG). The measurement of molecular diffusion is then used to extract information on the average properties of subcellular nanostructures, such as macromolecular crowding or molecular aggregation. Concerning the lysosome, fast RICS on a fluorescent tracer allowed us to quantitatively assess the increase in organelle viscosity in the pathological condition of Krabbe disease. In the case of ISGs, fast RICS on two ISG-specific secreting peptides unveiled their differential aggregation propensity depending on intragranular concentration. Finally, a combination of fast RICS and feedback-based 3D orbital tracking was used to subtract the slow movement of subcellular nanostructures from the fast diffusion of molecules contained within them and independently validate the results. Results presented here not only demonstrate the acquired ability to address the dynamic behavior of molecules in moving, nanoscopic reference systems, but prove the relevance of this approach to advance our knowledge on cell function at the subcellular scale.

Simulations of fluorescence fluctuation spectroscopy measurements of molecular diffusion in biomembranes

Studying the diffusion of individual molecules can yield important insights into the complex organization and function of the cell plasma membrane. Fluorescence fluctuation spectroscopy techniques allow the study of diffusion with single moleculesensitivity. Here, we present a novel, versatile and open accessible program based on Python for simulating molecular membrane diffusion. There have been different approaches to describe the diffusion laws governing the experimentally measured complex trajectories at the plasma membrane of live cells.

Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells.

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ranging from nanometers to micrometers and microseconds to minutes, respectively. Such a broad range of biological processes challenges conventional microscopy approaches. Here, we detail the procedure for implementing spot variation Fluorescence Correlation Spectroscopy (svFCS) measurements using a classical fluorescence microscope that has been customized. The protocol includes a specific performance check of the svFCS setup and the guidelines for molecular diffusion measurements by svFCS on the plasma membrane of living cells under physiological conditions. Additionally, we provide a procedure for disrupting plasma membrane raft nanodomains by cholesterol oxidase treatment and demonstrate how these changes in the lateral organization of the plasma membrane might be revealed by svFCS analysis. In conclusion, this fluorescence-based method can provide unprecedented details on the lateral organization of the plasma membrane with the appropriate spatial and temporal resolution.

Diffusion in nanopores: correlating experimental findings with \u201cfirst-principles\u201d predictions

Measurement of molecular diffusion in nanoporous host materials, which are typically inhomogeneous and anisotropic, often involves an intricate web of factors and relations to be taken into account since the associated diffusivities are a function of the diffusion path of the guest molecules during a given observation time. Depending on the observation time, therefore, the result of the experimental measurement can point to completely different conclusions about the underlying diffusion phenomena. The risk of misinterpretation of the experimental data, by correlating them with irrelevant phenomena, may be reduced if there is an option to compare the data with the results of totally independent measurements. The present communication addresses this issue with reference to the particular potentials of pulsed field gradient NMR and microimaging by infrared microscopy as techniques of microscopic diffusion measurement.

Probing Acetonitrile Solvation in Perfluorsulfonate Ion Exchange Membrane By in-Situ FTIR

In-situ FTIR is a successful tool to study water absorption and interactions within the Perfluorsulfonate Ion Exchange Membrane (PFSA)(1–4). The versatility of the technique can reveal in-depth information related to proton transport in PFSA, including proton dissociation and solvation. Another desirable feature is that experiments under controlled water content(λ) is also possible. Studies of PFSA membranes equilibrated with different λ was fundamental to understand membrane behavior(5). The volatile nature of acetonitrile and many organic solvents adds to the inaccuracy of many classic membrane characterization methods. To overcome this, techniques tailored for membrane incorporated with organic solvents are necessary. In this study, acetonitrile and lithiated PFSA were chosen to revisit the in-situ IR technique. The goal of this work is to examine acetonitrile’s role in membrane swelling and to create a ‘slow-motion picture’ of how acetonitrile molecules behave upon being imbibed into the membrane, especially at low λ. The vibrational signal of cyanide group (~2250 cm-1)does not overlap with other signals from the membrane and is known to respond to dipole interactions by shifting to higher wavenumber(6). This feature makes an acetonitrile-swollen system an even more favorable candidate for in-situ IR study compared to its aqueous counterpart. For studying proton transport in membrane, initial water buildup in membrane yields a weak and broad signal, making the already obscure -OH stretching region even more complicated to analyze. Without interference from hydrogen bonding, lithium ion solvation with cyanide can be clearly observed. Coupled with monitoring -SO3- area, it is totally feasible to simultaneously see both cation dissociation from membrane (Fig.1A) and cation solvation in solvent (Fig.1B) in a real-time manner to compare with fully swollen state. Acknowledgement We gratefully acknowledge the support of this work by the U.S. Department of Energy, Office of Electricity Delivery and Energy Reliability (Dr. Imre Gyuk). We also thank 3M for providing membranes. References Elabd YA, Baschetti MG, Barbari TA. Time-resolved Fourier transform infrared/attenuated total reflection spectroscopy for the measurement of molecular diffusion in polymers. J Polym Sci Part B Polym Phys. 2003;41(22):2794–807.Hallinan DT, Elabd YA. Diffusion of Water in Nafion Using Time-Resolved Fourier Transform Infrared−Attenuated Total Reflectance Spectroscopy. J Phys Chem B. 2009 ;113(13):4257–66.Hallinan DT, Elabd YA. Diffusion and Sorption of Methanol and Water in Nafion Using Time-Resolved Fourier Transform Infrared−Attenuated Total Reflectance Spectroscopy. J Phys Chem B. 2007 ;111(46):13221–30.Kunimatsu K, Bae B, Miyatake K, Uchida H, Watanabe M. ATR-FTIR Study of Water in Nafion Membrane Combined with Proton Conductivity Measurements during Hydration/Dehydration Cycle. J Phys Chem B. 2011 21;115(15):4315–21.Zawodzinski TA, Derouin C, Radzinski S, Sherman RJ, Smith VT, Springer TE, et al. Water Uptake by and Transport Through Nafion® 117 Membranes. J Electrochem Soc. 1993 ;140(4):1041–7.Barthel J, Buchner R, Wismeth E. FTIR Spectroscopy of Ion Solvation of LiClO4 and LiSCN in Acetonitrile, Benzonitrile, and Propylene Carbonate. J Solut Chem. 2000 ;29(10):937–54. Figure.1A: SO stretching shift due to solvation; Figure.1B: Normalized CN stretching area upon acetonitrile buildup in the membrane. Blue dashline indicates corresponding fully swollen state. Figure 1

Operando magnetic resonance studies of phase behaviour and oligomer accumulation within catalyst pores during heterogeneous catalytic ethene oligomerization

Two-dimensional 1H magnetic resonance imaging and spatially-resolved 1H magnetic resonance spectroscopy and diffusion measurements were recorded as a function of time-on-stream within a fixed-bed reactor to provide direct measurements of the progress of the heterogeneous catalytic oligomerization of ethene occurring over a 1 wt% Ni-Al2O3-SiO2 catalyst. The catalyst bed was of internal diameter 2 cm; magnetic resonance measurements were recorded over a bed length of 5.5 cm. Experiments were conducted at a temperature and pressure of 110 °C and 29 bara, respectively, with continuous downflow of ethene at a flowrate of 0.78 L h−1. During conversion the accumulation of 1H-containing species within the catalyst pellets was imaged, and spatially-resolved 1H NMR spectra were recorded at 1 mm intervals along the length of the reactor. Diffusion-filtered 1D chemical shift imaging was used to discriminate between gas- and liquid-phase species along the length of the reactor at 1 mm intervals. Finally, spectrally-encoded pulsed field gradient measurements of molecular diffusion were employed to infer the molecular composition of the gas and liquid phases and to identify populations of these phases inside and external to the pore space of the catalyst pellets; these measurements were spatially-resolved along the length of the reactor, with data being averaged over sections of height 4 mm. The results are consistent with oligomers of carbon number C20 and greater existing within the pores of the catalyst pellets which act to block the pore space, thereby deactivating the catalyst.

Role of Diffusion Tensor Imaging of the Median Nerve in Carpal Tunnel Syndrome: A Case Control Comparative Study with Electrophysiological Tests and Clinical Assessment

AbstractBackground: Carpal Tunnel Syndrome (CTS) is the most common peripheral neuropathy of an upper extremity. The diagnosis of CTS is commonly based on findings from the medical history, physical examination, and provocative tests. To confirm the diagnosis, some clinician accepts Electrophys-iological Studies (EPS) as the reference standard. Diffusion-Tensor Imaging (DTI) is a subtype of diffusion-weighted imaging based on measurement of molecular diffusion along multiple directions in space. Directional anisotropy of water diffusibility can be quantified by imaging indices such as Apparent Diffusion Coefficient (ADC) and Fractional Anisot-ropy (FA) used to characterize the degree of diffusion anisot-ropy.Aim of Work: The aim of this study is to study the efficacy of diffusion tensor imaging in the diagnosis of carpal tunnel syndrome.Material and Methods: Twenty-three wrists of 15 healthy subjects and 47 wrists of 31 CTS patients were evaluated with DTI and EPS. FA and ADC of the median nerve were calculated at four locations: Distal radioulnar joint, pisiform bone, hamate bone and hamate hook, and also determined from the whole median nerve. Electrophysiological test, including nerve conduction velocity was also performed for comparison with clinical assessment as a standard of reference.Results: There was a significant difference between healthy individuals and patients with carpal tunnel syndrome (p<0.01 for both FA and ADC). FA value less than 0.54 and ADC value more than 1.19 X 1 0–3mm2/sec might be used in the diagnosis of carpal tunnel syndrome. As regards the DTI results, the sensitivity, specificity, negative predictive value, positive predictive value and accuracy were 97.8%, 95.6%, 95.6%, 97.8% and 97.1% respectively.Conclusion: DTI scan contribute to carpal tunnel syndrome diagnosis on the basis of FA and ADC measurements.

Note: Molecular diffusivity in a small pore zeolite measured by a variable pressure (piezometric) uptake method.

The use of numerical analysis to solve the diffusion equation in the uptake method allowed the measurement of molecular diffusivity in a zeolite with a variable pressure around it. The diffusivity was obtained from the data in the measurement of the adsorption isotherm, which means that the diffusivity measurement now needs neither a special instrument nor procedure. The diffusivities of all the gases are readily available from the measurement of their adsorption isotherms and these data include how the diffusivity changes versus adsorbed concentration. The modeling introduced can also be used for a zeolite with a surface barrier.

Spatially Multiplexed Imaging: Fluorescence Correlation Spectroscopy for Efficient Measurement of Molecular Diffusion at Solid-Liquid Interfaces.

Fluorescence correlation spectroscopy (FCS) has become an important technique for the characterization of molecular dynamics, especially at interfaces. Fluorescence correlation spectroscopy provides both temporal and spatial resolution for measuring fast processes at equilibrium through analysis of noise in fluorescence intensities from the statistical fluctuations in a small number of molecules. The small molecular populations produce very low-level fluorescence signals, where time-averaging the fluorescence autocorrelation function is needed to generate reasonable signal-to-noise (S/N) ratios. Recently imaging cameras have been adapted to FCS measurements of molecular dynamics at interfaces (membranes and surfaces) through the use of electron-multiplying charge-coupled device (EM-CCD) detectors for acquisition of fluorescence from addressable areas on the detector. This approach provides a major advantage over traditional focused-spot FCS by allowing electronic control over the location and area of the acquired region on the sample surface. Imaging-FCS can also provide a spatial multiplexing advantage through its ability to measure intensity data from larger areas in parallel with no loss of time resolution. In this work, this multiplexing advantage is exploited to determine molecular diffusion rates from the simultaneous measurement of multiple areas on a surface, the autocorrelation traces from which are averaged to improve the S/N ratio. As proof of concept, the diffusion of 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI) on a C18-modified interface was measured using this multiplexed method and compared to autocorrelation data acquired from a single spot. Due to the slow thermal recovery of the EM-CCD that inhibits fast time-averaging, spatial multiplexing in imaging-FCS provides an eightyfold time savings to reach the same S/N ratio as multiple (time-averaged) measurements from a single spot.

Revealing mesoscopic structural universality with diffusion

Measuring molecular diffusion is widely used for characterizing materials and living organisms noninvasively. This characterization relies on relations between macroscopic diffusion metrics and structure at the mesoscopic scale commensurate with the diffusion length. Establishing such relations remains a fundamental challenge, hindering progress in materials science, porous media, and biomedical imaging. Here we show that the dynamical exponent in the time dependence of the diffusion coefficient distinguishes between the universality classes of the mesoscopic structural complexity. Our approach enables the interpretation of diffusion measurements by objectively selecting and modeling the most relevant structural features. As an example, the specific values of the dynamical exponent allow us to identify the relevant mesoscopic structure affecting MRI-measured water diffusion in muscles and in brain, and to elucidate the structural changes behind the decrease of diffusion coefficient in ischemic stroke.

Coaxial probe for nuclear magnetic resonance diffusion and relaxation correlation experiments

A coaxial nuclear magnetic resonance (NMR) probe is built to measure diffusion and relaxation properties of liquid samples. In particular, we demonstrate the acquisition of two-dimensional (2D) distribution functions (T1-T2 and diffusion–T2), essential for fluids characterization. The compact design holds promise for miniaturization, thus enabling the measurement of molecular diffusion that is inaccessible to conventional micro-NMR setups. Potential applications range from crude oil characterization to biomolecular screening and detections.

Value of the diffusion-weighted MRI in the differential diagnostics of malignant and benign kidney neoplasms - our clinical experience.

SummaryBackgroundDiffusion-weighted imaging (DWI) is an MRI modality using strong bipolar gradients to create a sensitivity of the signal to the thermally-induced Brownian motions of water molecules and in vivo measurement of molecular diffusion. The apparent diffusion coefficient (ADC) is a quantitative parameter calculated from DWI images which is used as a measure of diffusion. DWI allows to obtain comprehensive information on morphological and functional state of the kidney during a single examination without contrast medium administration. The purpose of the study was to evaluate the value of DWI in differentiating benign and malignant solid kidney tumors based on the initial stage of the study.Material/MethodsThe study included 19 adult patients with pathologically verified renal tumors: 9 patients with clear cell subtype of the renal cell carcinoma, 5 patients with oncocytoma and 5 patients with angiomyolipoma (AML). In addition, 5 healthy volunteers with completely normal findings according to kidney ultrasound were included into this study and set as reference. All patients underwent renal MR imaging which included DWI with subsequent ADC measurement. MR imaging was performed with a 1.5 T body scanner using an eight-channel phased-array body coil.ResultsThe mean ADC value of ccRCC was significantly lower than that of normal renal parenchyma (2.11±0.25×10−3 mm2/s vs. 3.36±0.41×10−3 mm2/s, p<0.01). There was a significant difference in ADC between the malignant and benign renal lesions: in patients with angiomyolipoma the ADC value was 2.36±0.32×10−3 mm2/s vs. 2.11±0.25×10−3 mm2/s; p<0.05 and in patients with oncocytoma – 2.75±0.27×10−3 mm2/s vs. 2.11±0.25×10−3 mm2/s; p<0.05. The difference in ADC values in patients with high and low ccRCC grades was observed.ConclusionsDWI can be used to characterize renal lesions; the ADC of a renal lesion can be potentially used as an additional parameter to help determine the appropriate clinical management.

Tackling Sample‐Related Artifacts in Membrane FCS Using Parallel SAF and UAF Detection

The complex shape and plasticity of cells is an intricate issue for the measurement of molecular diffusion in plasma membranes by fluorescence correlation spectroscopy (FCS). An important precondition for accurate diffusion measurements is a sufficient flatness of the membrane over the considered region and the absence of non-membrane-bound fluorescence diffusion. A method is presented to identify axial motion components caused by a non-ideal geometry of the membrane based on simultaneous measurement of the fluorescence emitted above and below the critical angle of the specimen/glass interface. Thereby, two detection volumes are generated that are laterally coincident, but differ in their axial penetration of the specimen. The similarity between the intensity tracks of the supercritical angle fluorescence (SAF) and the undercritical angle fluorescence (UAF) strongly depends on the membrane flatness and intracellular fluorescence, and can help to avoid sample-related artifacts in the diffusion measurement.

Transient diffraction grating measurements of molecular diffusion in the undergraduate laboratory

Diffusion is a central process in many biological, chemical, and physical systems. We describe an experiment that employs the interference of laser beams to allow the measurement of molecular diffusion on submillimeter length scales. The interference fringes of two intersecting pump beams within a dye solution create a sinusoidal distribution of long-lived molecular excited states. A third probe beam is incident at a wavelength at which the indices of refraction of the ground and excited states are different, so the probe beam diffracts from the spatially periodic excited-state pattern. After the pump beams are switched off, the excited-state periodicity washes out as the system diffuses back to equilibrium. The molecular diffusion constant is obtained from the rate constant of the exponential decay of the diffracted beam. It is also possible to measure the excited-state lifetime.

Recovery of Photoinduced Reversible Dark States Utilized for Molecular Diffusion Measurements

For a spatially restricted excitation volume, the effective modulation of the excitation in time is influenced by the passage times of the molecules through the excitation volume. By applying an additional time-modulated excitation, the buildup of photoinduced reversible dark states in fluorescent molecules can be made to vary significantly with their passage times through the excitation volume. The variations in the dark state populations are reflected by the time-averaged fluorescence intensity, which thus can be used to characterize the mobilities of the molecules. The concept was experimentally verified by measuring the fluorescence response of freely diffusing cyanine fluorophores (Cy5), undergoing trans-cis isomerization when subject to time-modulated excitation in a focused laser beam. From the fluorescence response, and by applying a simple photodynamic model, the transition times of the Cy5 molecules could be well reproduced when applying different laminar flow speeds through the detection volume. The presented approach puts no constraints on sample concentration, no requirements for high time resolution or sensitivity in the detection, nor requires a high fluorescence brightness of the characterized molecules. This can make the concept useful for a broad range of biomolecular mobility studies.

Post-Hoc Vibration Mitigation for Single-Molecule Tracking and Diffusion Measurements in Lipid Membranes

ABSTRACT Single-molecule tracking algorithms and analyses that use particle trajectory information rely on the accurate determination of particle positions to produce a meaningful interpretation of molecular dynamics. However, artifacts arising from vibration can sometimes distort or entirely mask the processes being studied. Here we describe a straightforward implementation of the Lucas-Kanade stabilization algorithm that can remove the impact of instrument-related vibrations on molecular diffusion measurements after a data set has been collected. Using fluorescently derivatized αHL on supported lipid bilayers in conjunction with computer simulations of 2D diffusion, we report that post-hoc stabilization can be effective in removing both simple and complex noise sources. The effectiveness of vibration mitigation is a function of the relationship between the vibration amplitude and the magnitude of the diffusion constant. For diffusion constants in the range of typical membrane proteins (e.g., 10−9 to 10−7 cm2s−1), vibrations that produce errors in excess of 100% can be reduced to producing errors less than 5%. The algorithm is effective even when the relative magnitude of the vibration is high and can be especially useful in experiments where vibrations cannot be entirely removed by experimental means alone. Supplemental materials are available for this article. Go to the publisher's online edition of Spectroscopy Letters for the following free supplemental resources: Video content illustrating single–molecule tracking capability, output from the single–molecule simulator, and the impact of post-hoc vibration stabilization.

Peak parking-moment analysis: A strategy for the measurement of molecular diffusivity in liquid phase

The peak-parking (PP) method permits the measurement of molecular diffusivities ( D m) in solutions. D m is first measured for benzene in pure methanol and acetonitrile (ACN), using an empty open tube. This yields an effective axial diffusion coefficient ( D ax,m) equal to D m because there is no tortuosity nor constriction in the flow channel. The same measurements made for the same combinations of solute and solvents, using a column packed with non-porous silica particles provides the obstructive factor ( Y m), defined as the ratio D ax,m/ D m, which accounts for the influence of tortuosity and constriction of the interparticulate space in packed columns on axial molecular dispersion. The value obtained for Y m, 0.74–0.75, is constant, irrespective of the solvent. Then, PP experiments were made with the same apparatus and column to measure D m for benzene, toluene, and ethylbenzene in aqueous solutions of methanol and ACN. The values of D m obtained by correcting the experimental D ax,m values with Y m were compared with values previously reported and those estimated by several literature correlations. They were in good agreement with each other. The average relative error is estimated at 4.5–10%, demonstrating that the PP method is practically effective for experimental measurements of D m.

생체 다공성 매질에서 분자 확산 측정을 위한 영상 기반 형광 광표백 기법 개발

Fluorescence recovery after photobleaching (FRAP) has been widely used for the measurement of molecular diffusion in living cells and tissues. We developed an image-based FRAP (iFRAP) technique using a modified real-time microscope and a 488 nm Ar-ion laser. A fractional intensity curve was obtained from the time-lapse images of fluorescence recovery in the bleached spot to determine the diffusion coefficient of fluorescently labeled macromolecules in porous medium. We validated iFRAP through experiments with agar gels (0.5% and 1.5% w/v) containing FITC-Dextrans (10, 70 and 500 kDa MW). Further validation was performed by a Monte Carlo approach, where we simulated the three-dimensional random walk of macromolecules in agar gel model. Diffusion coefficients were deduced from the mean square displacement curves and showed good agreements with those measured by iFRAP.

Peak parking method for measurement of molecular diffusivity in liquid phase systems

The combination of series of measurements of band broadening made with the peak parking (PP) method, using successively an open capillary tube and a HPLC column, gives a convenient procedure for the measurement of the molecular diffusivity ( D m) of compounds in solutions, of their axial dispersion coefficient ( D ax,m) in chromatographic columns, and of the tortuosity or obstructive factor of the column bed. The molecular diffusivity measured for benzene in methanol was in excellent agreement with literature data. The ratio of the axial dispersion coefficient to this diffusivity gives the obstructive factor ( γ m) of the packed bed, which was 0.74 for the column used. The values of D m in other solutions were obtained from the D ax,m values measured by the PP method, by correcting the D ax,m values with the γ m value. The D m values determined by this method were in good agreement with those previously reported or estimated using literature correlations. These results showed that the PP method is effective for the experimental measurement of D m.

Improved diffusion measurement in heterogeneous systems using the magic asymmetric gradient stimulated echo (MAGSTE) technique

A magic asymmetric gradient stimulated echo (MAGSTE) sequence was recently proposed to improve molecular diffusion measurements in the presence of spatially varying background gradients. Its effectiveness has been demonstrated previously with simulated background gradients and in phantoms that contain bulk susceptibility differences. In this study, we investigated the MAGSTE technique in microscopically heterogeneous systems, and compared it with the conventional bipolar pulsed gradient stimulated echo (bPGSTE) sequence. We demonstrated that the MASGTE measurements, compared to the bPGSTE method, varied significantly less when the diffusion encoding/decoding interval ( δ) was changed. In addition, the MAGSTE technique provided good characterization of the surface area-to-volume ratio for heterogeneous systems investigated in this study. In sum, this study showed that the MAGSTE technique provided diffusion measurements superior to those of the bPGSTE sequence, especially in the presence of severe heterogeneous background gradients.